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cd133 pe  (Miltenyi Biotec)


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    Miltenyi Biotec cd133 pe
    Spheroid formation, maturation, and functional CSC enrichment in HGSOC cell lines cultured under ultra‐low attachment (ULA) conditions. Representative brightfield microscopy images of OVCAR‐3 and OVSAHO cell lines cultured in ULA plates at relevant time points (4× objective). Yellow arrows indicate highly compartmentalized OVCAR‐3 spheroids, whereas red arrows denote overcrowded aggregates observed at extended culture durations (a). Schematic illustration summarizing spheroid morphologies observed under ULA conditions, including loose aggregates, compact spheroids, and compartmentalized spheroids (b). Quantitative analysis of spheroid diameter distribution and representative size comparison of highly compartmentalized OVCAR‐3 spheroids illustrating intra‐well heterogeneity within ULA cultures at Day 48 (10× objective) (c). Representative confocal microscopy images of an OVCAR‐3 spheroid at Day 48 including brightfield image, confocal z‐sections acquired at ∼2 µm intervals, and corresponding z‐stack projection (63× objective). Blue = DAPI (nuclei), green = α‐tubulin. Individual channels are shown separately to facilitate visualization of marker <t>distribution.</t> <t>CD133‐PE</t> monolayer negative controls and uncropped confocal images are provided in Figure S1 (d). Representative confocal microscopy images of additional aggregates and spheroids from the same culture (63X objective). Blue = DAPI, green = α‐tubulin, red = CD133‐PE. Individual channels are shown separately. CD133‐PE monolayer negative controls and uncropped images are provided in Figure (e). Growth inhibition curves and corresponding IC50 bar graphs for carboplatin, niraparib, paclitaxel, olaparib, cisplatin, and doxorubicin comparing OVCAR‐3 monolayer cultures (black bars) and ULA spheroids (gray bars). Viability was assessed using the NCI‐SRB assay (f). Data are presented as mean ± SD from three independent biological replicates. For SRB assays, each biological replicate contained technical triplicates. Statistical significance was determined using unpaired two‐tailed Student's t ‐tests or one‐way ANOVA where appropriate. * p < 0.05, ** p < 0.01, *** p < 0.001.
    Cd133 Pe, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 94/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/human+cd133/CD133%2F1+Antibody%2C+anti-human%2C+REAfinity/pmc13420801-103-0-1
    Average 94 stars, based on 44 article reviews
    cd133 pe - by Bioz Stars, 2026-09
    94/100 stars

    Images

    1) Product Images from "Benchmarking Ultra‐Low Attachment and Photopatterned GelMA 3D Culture Platforms for Modeling Cancer Stemness in High‐Grade Serous Ovarian Cancer"

    Article Title: Benchmarking Ultra‐Low Attachment and Photopatterned GelMA 3D Culture Platforms for Modeling Cancer Stemness in High‐Grade Serous Ovarian Cancer

    Journal: Biotechnology Journal

    doi: 10.1002/biot.70287

    Spheroid formation, maturation, and functional CSC enrichment in HGSOC cell lines cultured under ultra‐low attachment (ULA) conditions. Representative brightfield microscopy images of OVCAR‐3 and OVSAHO cell lines cultured in ULA plates at relevant time points (4× objective). Yellow arrows indicate highly compartmentalized OVCAR‐3 spheroids, whereas red arrows denote overcrowded aggregates observed at extended culture durations (a). Schematic illustration summarizing spheroid morphologies observed under ULA conditions, including loose aggregates, compact spheroids, and compartmentalized spheroids (b). Quantitative analysis of spheroid diameter distribution and representative size comparison of highly compartmentalized OVCAR‐3 spheroids illustrating intra‐well heterogeneity within ULA cultures at Day 48 (10× objective) (c). Representative confocal microscopy images of an OVCAR‐3 spheroid at Day 48 including brightfield image, confocal z‐sections acquired at ∼2 µm intervals, and corresponding z‐stack projection (63× objective). Blue = DAPI (nuclei), green = α‐tubulin. Individual channels are shown separately to facilitate visualization of marker distribution. CD133‐PE monolayer negative controls and uncropped confocal images are provided in Figure S1 (d). Representative confocal microscopy images of additional aggregates and spheroids from the same culture (63X objective). Blue = DAPI, green = α‐tubulin, red = CD133‐PE. Individual channels are shown separately. CD133‐PE monolayer negative controls and uncropped images are provided in Figure (e). Growth inhibition curves and corresponding IC50 bar graphs for carboplatin, niraparib, paclitaxel, olaparib, cisplatin, and doxorubicin comparing OVCAR‐3 monolayer cultures (black bars) and ULA spheroids (gray bars). Viability was assessed using the NCI‐SRB assay (f). Data are presented as mean ± SD from three independent biological replicates. For SRB assays, each biological replicate contained technical triplicates. Statistical significance was determined using unpaired two‐tailed Student's t ‐tests or one‐way ANOVA where appropriate. * p < 0.05, ** p < 0.01, *** p < 0.001.
    Figure Legend Snippet: Spheroid formation, maturation, and functional CSC enrichment in HGSOC cell lines cultured under ultra‐low attachment (ULA) conditions. Representative brightfield microscopy images of OVCAR‐3 and OVSAHO cell lines cultured in ULA plates at relevant time points (4× objective). Yellow arrows indicate highly compartmentalized OVCAR‐3 spheroids, whereas red arrows denote overcrowded aggregates observed at extended culture durations (a). Schematic illustration summarizing spheroid morphologies observed under ULA conditions, including loose aggregates, compact spheroids, and compartmentalized spheroids (b). Quantitative analysis of spheroid diameter distribution and representative size comparison of highly compartmentalized OVCAR‐3 spheroids illustrating intra‐well heterogeneity within ULA cultures at Day 48 (10× objective) (c). Representative confocal microscopy images of an OVCAR‐3 spheroid at Day 48 including brightfield image, confocal z‐sections acquired at ∼2 µm intervals, and corresponding z‐stack projection (63× objective). Blue = DAPI (nuclei), green = α‐tubulin. Individual channels are shown separately to facilitate visualization of marker distribution. CD133‐PE monolayer negative controls and uncropped confocal images are provided in Figure S1 (d). Representative confocal microscopy images of additional aggregates and spheroids from the same culture (63X objective). Blue = DAPI, green = α‐tubulin, red = CD133‐PE. Individual channels are shown separately. CD133‐PE monolayer negative controls and uncropped images are provided in Figure (e). Growth inhibition curves and corresponding IC50 bar graphs for carboplatin, niraparib, paclitaxel, olaparib, cisplatin, and doxorubicin comparing OVCAR‐3 monolayer cultures (black bars) and ULA spheroids (gray bars). Viability was assessed using the NCI‐SRB assay (f). Data are presented as mean ± SD from three independent biological replicates. For SRB assays, each biological replicate contained technical triplicates. Statistical significance was determined using unpaired two‐tailed Student's t ‐tests or one‐way ANOVA where appropriate. * p < 0.05, ** p < 0.01, *** p < 0.001.

    Techniques Used: Functional Assay, Cell Culture, Microscopy, Comparison, Confocal Microscopy, Marker, Inhibition, Sulforhodamine B Assay, Two Tailed Test

    Comparative benchmarking of CSC and EMT‐associated traits in HGSOC cells cultured using ULA and photopatterned GelMA platforms. Representative western blot images and corresponding quantitative bar graphs showing expression of CSC‐associated proteins SOX‐2, ALDH1A1, NANOG, and OCT‐4 in OVCAR‐3 and OVSAHO cultures. Protein levels were normalized to Vinculin and Calnexin, with representative loading control bands shown. Different loading controls were selected depending on target abundance, membrane compatibility, and subcellular localization. Quantification was performed using ImageJ (a). Representative flow cytometry pseudo‐dot plots with gates defined using unstained controls, and bar graphs summarizing the percentage of CD133‐PE, CD44‐FITC, CD117‐PE, and CXCR4‐FITC positive populations. Representative gating strategy is provided in Figure (b). Representative western blot images and quantitative bar graphs of EMT‐associated proteins Slug, Snail, TWIST1, vimentin, and fibronectin in OVCAR‐3 cultures (normalized to calnexin). E‐cadherin and N‐cadherin were additionally analyzed (normalized to α‐tubulin), with quantitative N‐cadherin/E‐cadherin ratios shown (c). Representative western blot analysis of stemness‐associated proteins in OVCAR‐3 cells cultured as 2D monolayer, 2D monolayer in spheroid enrichment medium, UV‐treated monolayer, ULA spheroids, and photopatterned GelMA (GelMA‐Pm) spheroids. Due to limited cell yield obtained from GelMA‐Pm cultures, only SOX‐2 and ALDH1A1 expression could be evaluated in these samples. Protein expression was normalized to GAPDH. Corresponding densitometric quantification is shown on the right. Full‐length uncropped blots are provided in Figure . (d). RT‐qPCR analysis of the stemness‐associated genes SOX‐2, NANOG, ALDH1A1, and c‐MYC in OVCAR‐3 cells cultured under 2D monolayer, 2D + sphere medium, 2D + UV, photopatterned GelMA (GelMA‐Pm), and ULA spheroid conditions. Gene expression levels were normalized to β‐actin and are presented relative to 2D monolayer controls (set to 1). Values above 1 indicate increased expression relative to monolayer cultures. Data represent mean ± SD from three independent biological replicates ( n = 3). Statistical significance was determined using multiple t ‐tests; significance symbols are defined as follows: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***) (e). Data are presented as mean ± SD from three independent biological replicates. Statistical significance was determined using unpaired two‐tailed Student's t ‐tests or one‐way ANOVA where appropriate. * p < 0.05, ** p < 0.01, *** p < 0.001. Western blot images were cropped for clarity; non‐adjacent lanes from the same membrane are indicated by spaces. Full‐length blots are provided in Figure .
    Figure Legend Snippet: Comparative benchmarking of CSC and EMT‐associated traits in HGSOC cells cultured using ULA and photopatterned GelMA platforms. Representative western blot images and corresponding quantitative bar graphs showing expression of CSC‐associated proteins SOX‐2, ALDH1A1, NANOG, and OCT‐4 in OVCAR‐3 and OVSAHO cultures. Protein levels were normalized to Vinculin and Calnexin, with representative loading control bands shown. Different loading controls were selected depending on target abundance, membrane compatibility, and subcellular localization. Quantification was performed using ImageJ (a). Representative flow cytometry pseudo‐dot plots with gates defined using unstained controls, and bar graphs summarizing the percentage of CD133‐PE, CD44‐FITC, CD117‐PE, and CXCR4‐FITC positive populations. Representative gating strategy is provided in Figure (b). Representative western blot images and quantitative bar graphs of EMT‐associated proteins Slug, Snail, TWIST1, vimentin, and fibronectin in OVCAR‐3 cultures (normalized to calnexin). E‐cadherin and N‐cadherin were additionally analyzed (normalized to α‐tubulin), with quantitative N‐cadherin/E‐cadherin ratios shown (c). Representative western blot analysis of stemness‐associated proteins in OVCAR‐3 cells cultured as 2D monolayer, 2D monolayer in spheroid enrichment medium, UV‐treated monolayer, ULA spheroids, and photopatterned GelMA (GelMA‐Pm) spheroids. Due to limited cell yield obtained from GelMA‐Pm cultures, only SOX‐2 and ALDH1A1 expression could be evaluated in these samples. Protein expression was normalized to GAPDH. Corresponding densitometric quantification is shown on the right. Full‐length uncropped blots are provided in Figure . (d). RT‐qPCR analysis of the stemness‐associated genes SOX‐2, NANOG, ALDH1A1, and c‐MYC in OVCAR‐3 cells cultured under 2D monolayer, 2D + sphere medium, 2D + UV, photopatterned GelMA (GelMA‐Pm), and ULA spheroid conditions. Gene expression levels were normalized to β‐actin and are presented relative to 2D monolayer controls (set to 1). Values above 1 indicate increased expression relative to monolayer cultures. Data represent mean ± SD from three independent biological replicates ( n = 3). Statistical significance was determined using multiple t ‐tests; significance symbols are defined as follows: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***) (e). Data are presented as mean ± SD from three independent biological replicates. Statistical significance was determined using unpaired two‐tailed Student's t ‐tests or one‐way ANOVA where appropriate. * p < 0.05, ** p < 0.01, *** p < 0.001. Western blot images were cropped for clarity; non‐adjacent lanes from the same membrane are indicated by spaces. Full‐length blots are provided in Figure .

    Techniques Used: Cell Culture, Western Blot, Expressing, Control, Membrane, Flow Cytometry, Quantitative RT-PCR, Gene Expression, Two Tailed Test

    Optimization of photopatterned GelMA hydrogels for spheroid formation and CSC enrichment in HGSOC cell lines. Representative brightfield microscopy images of photopatterned GelMA (GelMA‐Pm) cultures of OVCAR‐3 and OVSAHO cells (upper panels: 4× objective; lower panels: 10× objective), showing spheroid formation under optimized conditions (a). Schematic overview of the GelMA‐Pm fabrication workflow and representative image of square photomask‐generated polymerized GelMA structures visible to the naked eye (b). Quantitative comparison of spheroid number and spheroid diameter per mm 2 between ULA and GelMA‐Pm cultures (c). Representative brightfield microscopy images illustrating approximate spheroid diameter distributions within photopatterned GelMA cultures at Day 14, including representative OVCAR‐3 spheroids (∼100 µm) and smaller OVSAHO spheroids (∼50 µm) (d). Representative confocal microscopy images of GelMA hydrogels containing spheroids from OVCAR‐3 and OVSAHO cultures, demonstrating cell viability and spatial distribution (10× objective, with selected regions magnified to 20X as indicated by yellow boxes). Blue = DAPI (nuclei), green = α‐tubulin, red = CD133‐PE. Corresponding brightfield and fluorescence images of identical fields are shown (e). Data are presented as mean ± SD from three independent biological replicates. Statistical significance was determined using unpaired two‐tailed Student's t‐tests or one‐way ANOVA where appropriate. * p < 0.05, ** p < 0.01, *** p < 0.001.
    Figure Legend Snippet: Optimization of photopatterned GelMA hydrogels for spheroid formation and CSC enrichment in HGSOC cell lines. Representative brightfield microscopy images of photopatterned GelMA (GelMA‐Pm) cultures of OVCAR‐3 and OVSAHO cells (upper panels: 4× objective; lower panels: 10× objective), showing spheroid formation under optimized conditions (a). Schematic overview of the GelMA‐Pm fabrication workflow and representative image of square photomask‐generated polymerized GelMA structures visible to the naked eye (b). Quantitative comparison of spheroid number and spheroid diameter per mm 2 between ULA and GelMA‐Pm cultures (c). Representative brightfield microscopy images illustrating approximate spheroid diameter distributions within photopatterned GelMA cultures at Day 14, including representative OVCAR‐3 spheroids (∼100 µm) and smaller OVSAHO spheroids (∼50 µm) (d). Representative confocal microscopy images of GelMA hydrogels containing spheroids from OVCAR‐3 and OVSAHO cultures, demonstrating cell viability and spatial distribution (10× objective, with selected regions magnified to 20X as indicated by yellow boxes). Blue = DAPI (nuclei), green = α‐tubulin, red = CD133‐PE. Corresponding brightfield and fluorescence images of identical fields are shown (e). Data are presented as mean ± SD from three independent biological replicates. Statistical significance was determined using unpaired two‐tailed Student's t‐tests or one‐way ANOVA where appropriate. * p < 0.05, ** p < 0.01, *** p < 0.001.

    Techniques Used: Microscopy, Generated, Comparison, Confocal Microscopy, Fluorescence, Two Tailed Test

    Related Articles

    Staining:

    Article Title: Chemotherapy Enriches for Proinflammatory Macrophage Phenotypes that Support Cancer Stem-Like Cells and Disease Progression in Ovarian Cancer
    Article Snippet: .. Briefly, cells were stained for human CD117 (1:50, Miltenyi Biotec; Cat. # 130-111-593, RRID: AB_2654579) and human CD133 (1:50, Miltenyi Biotec; Cat. # 130-110-962, RRID: AB_2654888), and ALDH activity was analyzed via ALDEFLUOR Kit following manufacturer’s instructions (STEMCELL Technologies; Cat. # 01700). .. Cells were collected from in vitro samples and treated with Fc receptor blocker (Miltenyi Biotec; Cat# 130-059-901, RRID: AB_2892112) for 10 minutes in 2°C to 8°C followed by antibody staining for M1: hCD80-APC (1:20, BioLegend; Cat. # 305220, RRID: AB_2076147), for M2: hCD206-AlexaFluor488 (1:20, BioLegend; Cat. # 321114, RRID: AB_571875), and for viability SYTOX Blue (Thermo Fisher Scientific; Cat. # S34857).

    Article Title: Hyaluronic acid-functionalized gelatin hydrogels reveal extracellular matrix signals temper the efficacy of erlotinib against patient-derived glioblastoma specimens
    Article Snippet: .. Patient-derived GBM6 and GBM12 cells were stained with human-CD133 (PE conjugated 1:11 dilution; Miltenyi Biotec), then with human-CD68 (FITC conjugated 1:20 dilution; Invitrogen), following manufacturer’s protocol. ..

    Article Title: New Peptide-Drug Conjugates for Precise Targeting of SORT1-Mediated Vasculogenic Mimicry in the Tumor Microenvironment of TNBC-Derived MDA-MB-231 Breast and Ovarian ES-2 Clear Cell Carcinoma Cells
    Article Snippet: .. For detection of VM-associated structures, formalin-fixed, paraffin-embedded tumor tissue sections were stained with primary antibodies against mouse CD31 (#CM303A, Biocare Medical, Pacheco, CA), human CD133 (#130-090-422, Miltenyi Biotec, San Diego, CA) and human sortilin (#MABN1792, EMD Millipore Corporation, Temecula, CA). ..

    Article Title: Chemotherapy enriches for pro-inflammatory macrophage phenotypes that support cancer stem-like cells and disease progression in ovarian cancer
    Article Snippet: .. Briefly, cells were stained for human CD117 (1:50, Miltenyi Biotec; 158 D ow nloaded from http://aacrjournals.org/cancerrescom m un/article-pdf/doi/10.1158/2767-9764.C R C -24-0311/3498153/crc-24-0311.pdf by guest on 24 Septem ber 2024 11 Cat.# 130-111-593, RRID:AB_2654579) human CD133 (1:50, Miltenyi Biotec; Cat.# 130-110-159 962, RRID:AB_2654888), and ALDH activity was analyzed via ALDEFLUOR Kit following 160 manufacturer’s instructions (STEMCELL Technologies; Cat.# 01700). .. 161 Cells were collected from in vitro samples and treated with Fc receptor blocker (Miltenyi 162 Biotec; Cat# 130-059-901, RRID:AB_2892112) for 10 minutes in 2-8°C followed by antibody 163 staining (M1: hCD80-APC (1:20, BioLegend; Cat.# 305220, RRID:AB_2076147), M2: 164 hCD206-AlexaFluor488 (1:20, BioLegend; Cat.# 321114, RRID:AB_571875), and viability 165 Sytox Blue, ThermoFisher; Cat.# S34857).

    Article Title: Targeted liposomes
    Article Snippet: After weighing the tumors, single-cell suspensions were obtained from the tumors by collagenase digestion in Hank's balanced solution containing 1 mg/mL collagenase (Roche) and 2 mmol/L DNase I (Sigma) 1 h at 37° C. The fractionated cells were passed through a 70-μm cell strainer (Fisher Scientific, Pittsburgh, Pa.) and washed with PBS. .. To determine the level of apoptosis, single cells were stained with antibodies for cleaved caspase-3 (Cell Signaling Technology, Danvers, Mass.), and human CD133 (Miltenyi Biotec). .. The labeled cells were analyzed by flow-activated cell sorting (FACS) on BD FACS Aria flow cytometer (BD Biosciences, San Jose, Calif.).

    Activity Assay:

    Article Title: Chemotherapy Enriches for Proinflammatory Macrophage Phenotypes that Support Cancer Stem-Like Cells and Disease Progression in Ovarian Cancer
    Article Snippet: .. Briefly, cells were stained for human CD117 (1:50, Miltenyi Biotec; Cat. # 130-111-593, RRID: AB_2654579) and human CD133 (1:50, Miltenyi Biotec; Cat. # 130-110-962, RRID: AB_2654888), and ALDH activity was analyzed via ALDEFLUOR Kit following manufacturer’s instructions (STEMCELL Technologies; Cat. # 01700). .. Cells were collected from in vitro samples and treated with Fc receptor blocker (Miltenyi Biotec; Cat# 130-059-901, RRID: AB_2892112) for 10 minutes in 2°C to 8°C followed by antibody staining for M1: hCD80-APC (1:20, BioLegend; Cat. # 305220, RRID: AB_2076147), for M2: hCD206-AlexaFluor488 (1:20, BioLegend; Cat. # 321114, RRID: AB_571875), and for viability SYTOX Blue (Thermo Fisher Scientific; Cat. # S34857).

    Article Title: Chemotherapy enriches for pro-inflammatory macrophage phenotypes that support cancer stem-like cells and disease progression in ovarian cancer
    Article Snippet: .. Briefly, cells were stained for human CD117 (1:50, Miltenyi Biotec; 158 D ow nloaded from http://aacrjournals.org/cancerrescom m un/article-pdf/doi/10.1158/2767-9764.C R C -24-0311/3498153/crc-24-0311.pdf by guest on 24 Septem ber 2024 11 Cat.# 130-111-593, RRID:AB_2654579) human CD133 (1:50, Miltenyi Biotec; Cat.# 130-110-159 962, RRID:AB_2654888), and ALDH activity was analyzed via ALDEFLUOR Kit following 160 manufacturer’s instructions (STEMCELL Technologies; Cat.# 01700). .. 161 Cells were collected from in vitro samples and treated with Fc receptor blocker (Miltenyi 162 Biotec; Cat# 130-059-901, RRID:AB_2892112) for 10 minutes in 2-8°C followed by antibody 163 staining (M1: hCD80-APC (1:20, BioLegend; Cat.# 305220, RRID:AB_2076147), M2: 164 hCD206-AlexaFluor488 (1:20, BioLegend; Cat.# 321114, RRID:AB_571875), and viability 165 Sytox Blue, ThermoFisher; Cat.# S34857).

    Western Blot:

    Article Title: CD133⁺ Vesicles Mediate Resistance to RAS-ERK Inhibition Regulated by YAP Activation in Liver Cancer Cells
    Article Snippet: CD133 knockout mice (B6N;129S-Prom1 tm1(cre/ERT2)Gilb/ J) were purchased from the Jackson Laboratory and breed back with C57BL/6 wild type mice to C57BL/6 genetic background ( ). .. Proteins were extracted in RIPA buffer, and immunoblotting was performed using standard protocols as previously described., specific primary antibodies used in the experiments are listed here. mouse CD133 (14133182, eBioscience), human CD133 (86781, CST), human CD133 (130092395, miltenyi), CD133 (18470, proteintech), CD133 (66666, proteintech), β-Tubulin (66240, proteintech), pERK1/2 (4370, CST), ERK1/2 (4695, CST), pYAP (13008, CST), YAP (14074, CST), CD63 (GTX41875, GeneTex), Rab11 (5589, CST), Calnexin (2679, CST), VDAC1 (14734, abcam), H3 (4620, CST), Rab5A (46449, CST), EGFR (4267, CST), LAMP1 (21997, proteintech), Golgin-97 (13192, CST), Cyr61 (PA5-78022, Invitrogen) and Golgi58 (ab27043, abcam). .. Images are visualized using the ECL system (34580, Thermo Fisher) followed by Bio-Rad ChemiDoc imaging system.

    Formalin-fixed Paraffin-Embedded:

    Article Title: New Peptide-Drug Conjugates for Precise Targeting of SORT1-Mediated Vasculogenic Mimicry in the Tumor Microenvironment of TNBC-Derived MDA-MB-231 Breast and Ovarian ES-2 Clear Cell Carcinoma Cells
    Article Snippet: .. For detection of VM-associated structures, formalin-fixed, paraffin-embedded tumor tissue sections were stained with primary antibodies against mouse CD31 (#CM303A, Biocare Medical, Pacheco, CA), human CD133 (#130-090-422, Miltenyi Biotec, San Diego, CA) and human sortilin (#MABN1792, EMD Millipore Corporation, Temecula, CA). ..



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    Mouse Anti Human Cd133 1 Vio Bright R667 Ab, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Spheroid formation, maturation, and functional CSC enrichment in HGSOC cell lines cultured under ultra‐low attachment (ULA) conditions. Representative brightfield microscopy images of OVCAR‐3 and OVSAHO cell lines cultured in ULA plates at relevant time points (4× objective). Yellow arrows indicate highly compartmentalized OVCAR‐3 spheroids, whereas red arrows denote overcrowded aggregates observed at extended culture durations (a). Schematic illustration summarizing spheroid morphologies observed under ULA conditions, including loose aggregates, compact spheroids, and compartmentalized spheroids (b). Quantitative analysis of spheroid diameter distribution and representative size comparison of highly compartmentalized OVCAR‐3 spheroids illustrating intra‐well heterogeneity within ULA cultures at Day 48 (10× objective) (c). Representative confocal microscopy images of an OVCAR‐3 spheroid at Day 48 including brightfield image, confocal z‐sections acquired at ∼2 µm intervals, and corresponding z‐stack projection (63× objective). Blue = DAPI (nuclei), green = α‐tubulin. Individual channels are shown separately to facilitate visualization of marker <t>distribution.</t> <t>CD133‐PE</t> monolayer negative controls and uncropped confocal images are provided in Figure S1 (d). Representative confocal microscopy images of additional aggregates and spheroids from the same culture (63X objective). Blue = DAPI, green = α‐tubulin, red = CD133‐PE. Individual channels are shown separately. CD133‐PE monolayer negative controls and uncropped images are provided in Figure (e). Growth inhibition curves and corresponding IC50 bar graphs for carboplatin, niraparib, paclitaxel, olaparib, cisplatin, and doxorubicin comparing OVCAR‐3 monolayer cultures (black bars) and ULA spheroids (gray bars). Viability was assessed using the NCI‐SRB assay (f). Data are presented as mean ± SD from three independent biological replicates. For SRB assays, each biological replicate contained technical triplicates. Statistical significance was determined using unpaired two‐tailed Student's t ‐tests or one‐way ANOVA where appropriate. * p < 0.05, ** p < 0.01, *** p < 0.001.
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    Spheroid formation, maturation, and functional CSC enrichment in HGSOC cell lines cultured under ultra‐low attachment (ULA) conditions. Representative brightfield microscopy images of OVCAR‐3 and OVSAHO cell lines cultured in ULA plates at relevant time points (4× objective). Yellow arrows indicate highly compartmentalized OVCAR‐3 spheroids, whereas red arrows denote overcrowded aggregates observed at extended culture durations (a). Schematic illustration summarizing spheroid morphologies observed under ULA conditions, including loose aggregates, compact spheroids, and compartmentalized spheroids (b). Quantitative analysis of spheroid diameter distribution and representative size comparison of highly compartmentalized OVCAR‐3 spheroids illustrating intra‐well heterogeneity within ULA cultures at Day 48 (10× objective) (c). Representative confocal microscopy images of an OVCAR‐3 spheroid at Day 48 including brightfield image, confocal z‐sections acquired at ∼2 µm intervals, and corresponding z‐stack projection (63× objective). Blue = DAPI (nuclei), green = α‐tubulin. Individual channels are shown separately to facilitate visualization of marker <t>distribution.</t> <t>CD133‐PE</t> monolayer negative controls and uncropped confocal images are provided in Figure S1 (d). Representative confocal microscopy images of additional aggregates and spheroids from the same culture (63X objective). Blue = DAPI, green = α‐tubulin, red = CD133‐PE. Individual channels are shown separately. CD133‐PE monolayer negative controls and uncropped images are provided in Figure (e). Growth inhibition curves and corresponding IC50 bar graphs for carboplatin, niraparib, paclitaxel, olaparib, cisplatin, and doxorubicin comparing OVCAR‐3 monolayer cultures (black bars) and ULA spheroids (gray bars). Viability was assessed using the NCI‐SRB assay (f). Data are presented as mean ± SD from three independent biological replicates. For SRB assays, each biological replicate contained technical triplicates. Statistical significance was determined using unpaired two‐tailed Student's t ‐tests or one‐way ANOVA where appropriate. * p < 0.05, ** p < 0.01, *** p < 0.001.
    Anti Cd133 Ntibody, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti cd133  (Miltenyi Biotec)
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    Spheroid formation, maturation, and functional CSC enrichment in HGSOC cell lines cultured under ultra‐low attachment (ULA) conditions. Representative brightfield microscopy images of OVCAR‐3 and OVSAHO cell lines cultured in ULA plates at relevant time points (4× objective). Yellow arrows indicate highly compartmentalized OVCAR‐3 spheroids, whereas red arrows denote overcrowded aggregates observed at extended culture durations (a). Schematic illustration summarizing spheroid morphologies observed under ULA conditions, including loose aggregates, compact spheroids, and compartmentalized spheroids (b). Quantitative analysis of spheroid diameter distribution and representative size comparison of highly compartmentalized OVCAR‐3 spheroids illustrating intra‐well heterogeneity within ULA cultures at Day 48 (10× objective) (c). Representative confocal microscopy images of an OVCAR‐3 spheroid at Day 48 including brightfield image, confocal z‐sections acquired at ∼2 µm intervals, and corresponding z‐stack projection (63× objective). Blue = DAPI (nuclei), green = α‐tubulin. Individual channels are shown separately to facilitate visualization of marker <t>distribution.</t> <t>CD133‐PE</t> monolayer negative controls and uncropped confocal images are provided in Figure S1 (d). Representative confocal microscopy images of additional aggregates and spheroids from the same culture (63X objective). Blue = DAPI, green = α‐tubulin, red = CD133‐PE. Individual channels are shown separately. CD133‐PE monolayer negative controls and uncropped images are provided in Figure (e). Growth inhibition curves and corresponding IC50 bar graphs for carboplatin, niraparib, paclitaxel, olaparib, cisplatin, and doxorubicin comparing OVCAR‐3 monolayer cultures (black bars) and ULA spheroids (gray bars). Viability was assessed using the NCI‐SRB assay (f). Data are presented as mean ± SD from three independent biological replicates. For SRB assays, each biological replicate contained technical triplicates. Statistical significance was determined using unpaired two‐tailed Student's t ‐tests or one‐way ANOVA where appropriate. * p < 0.05, ** p < 0.01, *** p < 0.001.
    Anti Cd133, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Serological parameters <t>and</t> <t>CD133/2</t> + sEVs in HCC and iCCA. (A) Overview of routinely assessed standard serum parameters in biliary and hepatic malignancies, including albumin, ALP, CA19-9, and CRP.(B) Platelet (thrombocyte) counts. (C) Total numbers of CD133/2 + sEVs and their CD9 + , CD63 + , and CD81 + subpopulations immobilized on Human ExoView® Tetraspanin Chips precoated with anti-CD9, anti-CD63, and anti-CD81 EV capture antibodies, including MIgG isotype controls. Bars represent median values with 95% CIs. Associated AUROC values, sensitivity, specificity, and cut-off values were calculated using GraphPad PRISM and subsequently applied in the analogous additive scoring system. Statistical analyses were performed using GraphPad PRISM (version 10.4.1). Group comparisons were conducted using two-sided Mann-Whitney U tests because of non-normal data distribution. Exact p values are shown and were as follows: albumin, p = 0.06; ALP, p <0.001; CA19-9, p = 0.03; and CRP, p <0.001 (A); thrombocytes, p = 0.002 (B); CD9 + CD133/2 + , p = 0.0014; CD63 + CD133/2 + , p = 0.0009; and CD81 + CD133/2 + , p = 0.0001 (C). Post-hoc effect size (Cohen’s d) and power analyses were derived from the Mann-Whitney U test results to quantify the discriminatory sensitivity of each biomarker comparison . ALP, alkaline phosphatase; CRP, C-reactive protein; EV, extracellular vesicle; HCC, hepatocellular carcinoma; iCCA, intrahepatic cholangiocarcinoma; MIgG, mouse IgG; sEV, small extracellular vesicle.
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    Serological parameters <t>and</t> <t>CD133/2</t> + sEVs in HCC and iCCA. (A) Overview of routinely assessed standard serum parameters in biliary and hepatic malignancies, including albumin, ALP, CA19-9, and CRP.(B) Platelet (thrombocyte) counts. (C) Total numbers of CD133/2 + sEVs and their CD9 + , CD63 + , and CD81 + subpopulations immobilized on Human ExoView® Tetraspanin Chips precoated with anti-CD9, anti-CD63, and anti-CD81 EV capture antibodies, including MIgG isotype controls. Bars represent median values with 95% CIs. Associated AUROC values, sensitivity, specificity, and cut-off values were calculated using GraphPad PRISM and subsequently applied in the analogous additive scoring system. Statistical analyses were performed using GraphPad PRISM (version 10.4.1). Group comparisons were conducted using two-sided Mann-Whitney U tests because of non-normal data distribution. Exact p values are shown and were as follows: albumin, p = 0.06; ALP, p <0.001; CA19-9, p = 0.03; and CRP, p <0.001 (A); thrombocytes, p = 0.002 (B); CD9 + CD133/2 + , p = 0.0014; CD63 + CD133/2 + , p = 0.0009; and CD81 + CD133/2 + , p = 0.0001 (C). Post-hoc effect size (Cohen’s d) and power analyses were derived from the Mann-Whitney U test results to quantify the discriminatory sensitivity of each biomarker comparison . ALP, alkaline phosphatase; CRP, C-reactive protein; EV, extracellular vesicle; HCC, hepatocellular carcinoma; iCCA, intrahepatic cholangiocarcinoma; MIgG, mouse IgG; sEV, small extracellular vesicle.
    Anti Cd133 1, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Positive and negative control cells for both marker panels. ( A ) HeLa (white arrows): in the first panel positive for cytokeratin (CK), Vimentin (Vim), <t>VEGF</t> and in the second panel positive for p16INK4A, negative for PD-L1 and CD45. ( B ) T98G (red arrows): positive for Vim. ( C ) CaSki (yellow arrows): positive for CK, Vim, VEGF, PD-L1 and p16INK4A. ( D ) MCF-7 (green arrows): positive for CK. All cell lines were negative for CD45. Only hematopoietic cells showed CD45 positive staining. Scale bar = 10 μm. Images were processed using Zeiss ZEN 3.7 software and the figure was assembled using Microsoft PowerPoint.
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    cd133  (Miltenyi Biotec)
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    Effect of MIT-A on stemness-associated markers in HCC cells. ( A ) Heatmap showing the expression of stemness-associated genes ( PROM1 <t>/CD133,</t> CD24 , MYC , ITGB1 , CD44 , EPCAM , KLF4 ) and direct SP1 targets ( SETDB1 , DNMT3A , DNMT3B ) in Huh7 WT and SR cells after MIT-A treatment. ( B ) RT-qPCR analysis of SP1, pluripotency markers ( NANOG , SOX2 , POU5F1 , KLF4 ), and surface markers ( CD24 , PROM1/ CD133 ) in Huh7 WT, Huh7 SR, and HepG2 cells treated with MIT-A (200 nM, 24 h for Huh7 WT and HepG2; 400 nM, 24 h for Huh7 SR). Expression was normalized to 18S. ( C ) Western blot analysis of stemness regulators (OCT4, NANOG, CD133), SP1, and SETDB1 in Huh7 WT and SR cells treated with MIT-A. β-actin served as loading control. Representative blots and quantifications are shown. ( D ) Flow cytometry analysis of CSC surface markers (CD24, CD133) in Huh7 WT, Huh7 SR, and HepG2 cells after MIT-A exposure. Representative histograms are shown for Huh7 WT cells (red, control; violet, MIT-A; blue, isotype). Mean fluorescence intensity (MFI) values are shown on the right. Quantification is presented as bar graphs. Data are presented as mean ± SEM from independent experiments (( B , C ): n = 3; ( D ): n = 4–6). Statistical analyses were performed using unpaired two-tailed Student’s t -tests ( B ) or Welch’s t -tests when appropriate ( C , D ). Statistical significance is indicated as * p < 0.05, ** p < 0.01, *** p < 0.001; NS, not significant.
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    Image Search Results


    Spheroid formation, maturation, and functional CSC enrichment in HGSOC cell lines cultured under ultra‐low attachment (ULA) conditions. Representative brightfield microscopy images of OVCAR‐3 and OVSAHO cell lines cultured in ULA plates at relevant time points (4× objective). Yellow arrows indicate highly compartmentalized OVCAR‐3 spheroids, whereas red arrows denote overcrowded aggregates observed at extended culture durations (a). Schematic illustration summarizing spheroid morphologies observed under ULA conditions, including loose aggregates, compact spheroids, and compartmentalized spheroids (b). Quantitative analysis of spheroid diameter distribution and representative size comparison of highly compartmentalized OVCAR‐3 spheroids illustrating intra‐well heterogeneity within ULA cultures at Day 48 (10× objective) (c). Representative confocal microscopy images of an OVCAR‐3 spheroid at Day 48 including brightfield image, confocal z‐sections acquired at ∼2 µm intervals, and corresponding z‐stack projection (63× objective). Blue = DAPI (nuclei), green = α‐tubulin. Individual channels are shown separately to facilitate visualization of marker distribution. CD133‐PE monolayer negative controls and uncropped confocal images are provided in Figure S1 (d). Representative confocal microscopy images of additional aggregates and spheroids from the same culture (63X objective). Blue = DAPI, green = α‐tubulin, red = CD133‐PE. Individual channels are shown separately. CD133‐PE monolayer negative controls and uncropped images are provided in Figure (e). Growth inhibition curves and corresponding IC50 bar graphs for carboplatin, niraparib, paclitaxel, olaparib, cisplatin, and doxorubicin comparing OVCAR‐3 monolayer cultures (black bars) and ULA spheroids (gray bars). Viability was assessed using the NCI‐SRB assay (f). Data are presented as mean ± SD from three independent biological replicates. For SRB assays, each biological replicate contained technical triplicates. Statistical significance was determined using unpaired two‐tailed Student's t ‐tests or one‐way ANOVA where appropriate. * p < 0.05, ** p < 0.01, *** p < 0.001.

    Journal: Biotechnology Journal

    Article Title: Benchmarking Ultra‐Low Attachment and Photopatterned GelMA 3D Culture Platforms for Modeling Cancer Stemness in High‐Grade Serous Ovarian Cancer

    doi: 10.1002/biot.70287

    Figure Lengend Snippet: Spheroid formation, maturation, and functional CSC enrichment in HGSOC cell lines cultured under ultra‐low attachment (ULA) conditions. Representative brightfield microscopy images of OVCAR‐3 and OVSAHO cell lines cultured in ULA plates at relevant time points (4× objective). Yellow arrows indicate highly compartmentalized OVCAR‐3 spheroids, whereas red arrows denote overcrowded aggregates observed at extended culture durations (a). Schematic illustration summarizing spheroid morphologies observed under ULA conditions, including loose aggregates, compact spheroids, and compartmentalized spheroids (b). Quantitative analysis of spheroid diameter distribution and representative size comparison of highly compartmentalized OVCAR‐3 spheroids illustrating intra‐well heterogeneity within ULA cultures at Day 48 (10× objective) (c). Representative confocal microscopy images of an OVCAR‐3 spheroid at Day 48 including brightfield image, confocal z‐sections acquired at ∼2 µm intervals, and corresponding z‐stack projection (63× objective). Blue = DAPI (nuclei), green = α‐tubulin. Individual channels are shown separately to facilitate visualization of marker distribution. CD133‐PE monolayer negative controls and uncropped confocal images are provided in Figure S1 (d). Representative confocal microscopy images of additional aggregates and spheroids from the same culture (63X objective). Blue = DAPI, green = α‐tubulin, red = CD133‐PE. Individual channels are shown separately. CD133‐PE monolayer negative controls and uncropped images are provided in Figure (e). Growth inhibition curves and corresponding IC50 bar graphs for carboplatin, niraparib, paclitaxel, olaparib, cisplatin, and doxorubicin comparing OVCAR‐3 monolayer cultures (black bars) and ULA spheroids (gray bars). Viability was assessed using the NCI‐SRB assay (f). Data are presented as mean ± SD from three independent biological replicates. For SRB assays, each biological replicate contained technical triplicates. Statistical significance was determined using unpaired two‐tailed Student's t ‐tests or one‐way ANOVA where appropriate. * p < 0.05, ** p < 0.01, *** p < 0.001.

    Article Snippet: CD133‐PE (Miltenyi Biotec, equal mixing of 130‐110‐962 and 130‐110‐962, 1:50–1:50 = 2:100) antibody was diluted 1:50 in CellO‐IF and incubation was done at 4°C for 20 min.

    Techniques: Functional Assay, Cell Culture, Microscopy, Comparison, Confocal Microscopy, Marker, Inhibition, Sulforhodamine B Assay, Two Tailed Test

    Comparative benchmarking of CSC and EMT‐associated traits in HGSOC cells cultured using ULA and photopatterned GelMA platforms. Representative western blot images and corresponding quantitative bar graphs showing expression of CSC‐associated proteins SOX‐2, ALDH1A1, NANOG, and OCT‐4 in OVCAR‐3 and OVSAHO cultures. Protein levels were normalized to Vinculin and Calnexin, with representative loading control bands shown. Different loading controls were selected depending on target abundance, membrane compatibility, and subcellular localization. Quantification was performed using ImageJ (a). Representative flow cytometry pseudo‐dot plots with gates defined using unstained controls, and bar graphs summarizing the percentage of CD133‐PE, CD44‐FITC, CD117‐PE, and CXCR4‐FITC positive populations. Representative gating strategy is provided in Figure (b). Representative western blot images and quantitative bar graphs of EMT‐associated proteins Slug, Snail, TWIST1, vimentin, and fibronectin in OVCAR‐3 cultures (normalized to calnexin). E‐cadherin and N‐cadherin were additionally analyzed (normalized to α‐tubulin), with quantitative N‐cadherin/E‐cadherin ratios shown (c). Representative western blot analysis of stemness‐associated proteins in OVCAR‐3 cells cultured as 2D monolayer, 2D monolayer in spheroid enrichment medium, UV‐treated monolayer, ULA spheroids, and photopatterned GelMA (GelMA‐Pm) spheroids. Due to limited cell yield obtained from GelMA‐Pm cultures, only SOX‐2 and ALDH1A1 expression could be evaluated in these samples. Protein expression was normalized to GAPDH. Corresponding densitometric quantification is shown on the right. Full‐length uncropped blots are provided in Figure . (d). RT‐qPCR analysis of the stemness‐associated genes SOX‐2, NANOG, ALDH1A1, and c‐MYC in OVCAR‐3 cells cultured under 2D monolayer, 2D + sphere medium, 2D + UV, photopatterned GelMA (GelMA‐Pm), and ULA spheroid conditions. Gene expression levels were normalized to β‐actin and are presented relative to 2D monolayer controls (set to 1). Values above 1 indicate increased expression relative to monolayer cultures. Data represent mean ± SD from three independent biological replicates ( n = 3). Statistical significance was determined using multiple t ‐tests; significance symbols are defined as follows: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***) (e). Data are presented as mean ± SD from three independent biological replicates. Statistical significance was determined using unpaired two‐tailed Student's t ‐tests or one‐way ANOVA where appropriate. * p < 0.05, ** p < 0.01, *** p < 0.001. Western blot images were cropped for clarity; non‐adjacent lanes from the same membrane are indicated by spaces. Full‐length blots are provided in Figure .

    Journal: Biotechnology Journal

    Article Title: Benchmarking Ultra‐Low Attachment and Photopatterned GelMA 3D Culture Platforms for Modeling Cancer Stemness in High‐Grade Serous Ovarian Cancer

    doi: 10.1002/biot.70287

    Figure Lengend Snippet: Comparative benchmarking of CSC and EMT‐associated traits in HGSOC cells cultured using ULA and photopatterned GelMA platforms. Representative western blot images and corresponding quantitative bar graphs showing expression of CSC‐associated proteins SOX‐2, ALDH1A1, NANOG, and OCT‐4 in OVCAR‐3 and OVSAHO cultures. Protein levels were normalized to Vinculin and Calnexin, with representative loading control bands shown. Different loading controls were selected depending on target abundance, membrane compatibility, and subcellular localization. Quantification was performed using ImageJ (a). Representative flow cytometry pseudo‐dot plots with gates defined using unstained controls, and bar graphs summarizing the percentage of CD133‐PE, CD44‐FITC, CD117‐PE, and CXCR4‐FITC positive populations. Representative gating strategy is provided in Figure (b). Representative western blot images and quantitative bar graphs of EMT‐associated proteins Slug, Snail, TWIST1, vimentin, and fibronectin in OVCAR‐3 cultures (normalized to calnexin). E‐cadherin and N‐cadherin were additionally analyzed (normalized to α‐tubulin), with quantitative N‐cadherin/E‐cadherin ratios shown (c). Representative western blot analysis of stemness‐associated proteins in OVCAR‐3 cells cultured as 2D monolayer, 2D monolayer in spheroid enrichment medium, UV‐treated monolayer, ULA spheroids, and photopatterned GelMA (GelMA‐Pm) spheroids. Due to limited cell yield obtained from GelMA‐Pm cultures, only SOX‐2 and ALDH1A1 expression could be evaluated in these samples. Protein expression was normalized to GAPDH. Corresponding densitometric quantification is shown on the right. Full‐length uncropped blots are provided in Figure . (d). RT‐qPCR analysis of the stemness‐associated genes SOX‐2, NANOG, ALDH1A1, and c‐MYC in OVCAR‐3 cells cultured under 2D monolayer, 2D + sphere medium, 2D + UV, photopatterned GelMA (GelMA‐Pm), and ULA spheroid conditions. Gene expression levels were normalized to β‐actin and are presented relative to 2D monolayer controls (set to 1). Values above 1 indicate increased expression relative to monolayer cultures. Data represent mean ± SD from three independent biological replicates ( n = 3). Statistical significance was determined using multiple t ‐tests; significance symbols are defined as follows: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***) (e). Data are presented as mean ± SD from three independent biological replicates. Statistical significance was determined using unpaired two‐tailed Student's t ‐tests or one‐way ANOVA where appropriate. * p < 0.05, ** p < 0.01, *** p < 0.001. Western blot images were cropped for clarity; non‐adjacent lanes from the same membrane are indicated by spaces. Full‐length blots are provided in Figure .

    Article Snippet: CD133‐PE (Miltenyi Biotec, equal mixing of 130‐110‐962 and 130‐110‐962, 1:50–1:50 = 2:100) antibody was diluted 1:50 in CellO‐IF and incubation was done at 4°C for 20 min.

    Techniques: Cell Culture, Western Blot, Expressing, Control, Membrane, Flow Cytometry, Quantitative RT-PCR, Gene Expression, Two Tailed Test

    Optimization of photopatterned GelMA hydrogels for spheroid formation and CSC enrichment in HGSOC cell lines. Representative brightfield microscopy images of photopatterned GelMA (GelMA‐Pm) cultures of OVCAR‐3 and OVSAHO cells (upper panels: 4× objective; lower panels: 10× objective), showing spheroid formation under optimized conditions (a). Schematic overview of the GelMA‐Pm fabrication workflow and representative image of square photomask‐generated polymerized GelMA structures visible to the naked eye (b). Quantitative comparison of spheroid number and spheroid diameter per mm 2 between ULA and GelMA‐Pm cultures (c). Representative brightfield microscopy images illustrating approximate spheroid diameter distributions within photopatterned GelMA cultures at Day 14, including representative OVCAR‐3 spheroids (∼100 µm) and smaller OVSAHO spheroids (∼50 µm) (d). Representative confocal microscopy images of GelMA hydrogels containing spheroids from OVCAR‐3 and OVSAHO cultures, demonstrating cell viability and spatial distribution (10× objective, with selected regions magnified to 20X as indicated by yellow boxes). Blue = DAPI (nuclei), green = α‐tubulin, red = CD133‐PE. Corresponding brightfield and fluorescence images of identical fields are shown (e). Data are presented as mean ± SD from three independent biological replicates. Statistical significance was determined using unpaired two‐tailed Student's t‐tests or one‐way ANOVA where appropriate. * p < 0.05, ** p < 0.01, *** p < 0.001.

    Journal: Biotechnology Journal

    Article Title: Benchmarking Ultra‐Low Attachment and Photopatterned GelMA 3D Culture Platforms for Modeling Cancer Stemness in High‐Grade Serous Ovarian Cancer

    doi: 10.1002/biot.70287

    Figure Lengend Snippet: Optimization of photopatterned GelMA hydrogels for spheroid formation and CSC enrichment in HGSOC cell lines. Representative brightfield microscopy images of photopatterned GelMA (GelMA‐Pm) cultures of OVCAR‐3 and OVSAHO cells (upper panels: 4× objective; lower panels: 10× objective), showing spheroid formation under optimized conditions (a). Schematic overview of the GelMA‐Pm fabrication workflow and representative image of square photomask‐generated polymerized GelMA structures visible to the naked eye (b). Quantitative comparison of spheroid number and spheroid diameter per mm 2 between ULA and GelMA‐Pm cultures (c). Representative brightfield microscopy images illustrating approximate spheroid diameter distributions within photopatterned GelMA cultures at Day 14, including representative OVCAR‐3 spheroids (∼100 µm) and smaller OVSAHO spheroids (∼50 µm) (d). Representative confocal microscopy images of GelMA hydrogels containing spheroids from OVCAR‐3 and OVSAHO cultures, demonstrating cell viability and spatial distribution (10× objective, with selected regions magnified to 20X as indicated by yellow boxes). Blue = DAPI (nuclei), green = α‐tubulin, red = CD133‐PE. Corresponding brightfield and fluorescence images of identical fields are shown (e). Data are presented as mean ± SD from three independent biological replicates. Statistical significance was determined using unpaired two‐tailed Student's t‐tests or one‐way ANOVA where appropriate. * p < 0.05, ** p < 0.01, *** p < 0.001.

    Article Snippet: CD133‐PE (Miltenyi Biotec, equal mixing of 130‐110‐962 and 130‐110‐962, 1:50–1:50 = 2:100) antibody was diluted 1:50 in CellO‐IF and incubation was done at 4°C for 20 min.

    Techniques: Microscopy, Generated, Comparison, Confocal Microscopy, Fluorescence, Two Tailed Test

    Serological parameters and CD133/2 + sEVs in HCC and iCCA. (A) Overview of routinely assessed standard serum parameters in biliary and hepatic malignancies, including albumin, ALP, CA19-9, and CRP.(B) Platelet (thrombocyte) counts. (C) Total numbers of CD133/2 + sEVs and their CD9 + , CD63 + , and CD81 + subpopulations immobilized on Human ExoView® Tetraspanin Chips precoated with anti-CD9, anti-CD63, and anti-CD81 EV capture antibodies, including MIgG isotype controls. Bars represent median values with 95% CIs. Associated AUROC values, sensitivity, specificity, and cut-off values were calculated using GraphPad PRISM and subsequently applied in the analogous additive scoring system. Statistical analyses were performed using GraphPad PRISM (version 10.4.1). Group comparisons were conducted using two-sided Mann-Whitney U tests because of non-normal data distribution. Exact p values are shown and were as follows: albumin, p = 0.06; ALP, p <0.001; CA19-9, p = 0.03; and CRP, p <0.001 (A); thrombocytes, p = 0.002 (B); CD9 + CD133/2 + , p = 0.0014; CD63 + CD133/2 + , p = 0.0009; and CD81 + CD133/2 + , p = 0.0001 (C). Post-hoc effect size (Cohen’s d) and power analyses were derived from the Mann-Whitney U test results to quantify the discriminatory sensitivity of each biomarker comparison . ALP, alkaline phosphatase; CRP, C-reactive protein; EV, extracellular vesicle; HCC, hepatocellular carcinoma; iCCA, intrahepatic cholangiocarcinoma; MIgG, mouse IgG; sEV, small extracellular vesicle.

    Journal: JHEP Reports

    Article Title: AI-guided additive scoring model for differential diagnosis of primary liver cancer

    doi: 10.1016/j.jhepr.2026.101826

    Figure Lengend Snippet: Serological parameters and CD133/2 + sEVs in HCC and iCCA. (A) Overview of routinely assessed standard serum parameters in biliary and hepatic malignancies, including albumin, ALP, CA19-9, and CRP.(B) Platelet (thrombocyte) counts. (C) Total numbers of CD133/2 + sEVs and their CD9 + , CD63 + , and CD81 + subpopulations immobilized on Human ExoView® Tetraspanin Chips precoated with anti-CD9, anti-CD63, and anti-CD81 EV capture antibodies, including MIgG isotype controls. Bars represent median values with 95% CIs. Associated AUROC values, sensitivity, specificity, and cut-off values were calculated using GraphPad PRISM and subsequently applied in the analogous additive scoring system. Statistical analyses were performed using GraphPad PRISM (version 10.4.1). Group comparisons were conducted using two-sided Mann-Whitney U tests because of non-normal data distribution. Exact p values are shown and were as follows: albumin, p = 0.06; ALP, p <0.001; CA19-9, p = 0.03; and CRP, p <0.001 (A); thrombocytes, p = 0.002 (B); CD9 + CD133/2 + , p = 0.0014; CD63 + CD133/2 + , p = 0.0009; and CD81 + CD133/2 + , p = 0.0001 (C). Post-hoc effect size (Cohen’s d) and power analyses were derived from the Mann-Whitney U test results to quantify the discriminatory sensitivity of each biomarker comparison . ALP, alkaline phosphatase; CRP, C-reactive protein; EV, extracellular vesicle; HCC, hepatocellular carcinoma; iCCA, intrahepatic cholangiocarcinoma; MIgG, mouse IgG; sEV, small extracellular vesicle.

    Article Snippet: Anti-CD133/2 antibodies (130-112-195, Miltenyi Biotec) and anti-CD44v6 antibodies (130-111-238, Miltenyi Biotec) were added and titrated against their matching isotype (REA293 phycoerythrin, 130-107-771 and REA293 allophycocyanin, 130-113-446, Miltenyi Biotec) and used in concentrations according to the respective antibodies; 7-AAD (BD Pharmingen, NJ, USA, 559925) was used for dead cell exclusion.

    Techniques: MANN-WHITNEY, Derivative Assay, Biomarker Discovery, Comparison

    Selection of the final marker set using LASSO and PCA. (A) The LASSO path plot illustrates the selection process of candidate markers using LASSO regression first. Early entrants, such as ALP, CRP, and CD9 + CD133/2 + small EVs, achieved high coefficient values, indicating consistent relevance within the fitted model. By contrast, thrombocytes, AFP, and albumin contributed with lower coefficients, whereas late entrants, including CA19-9 and CD63 + CD133/2 + small EVs, had the weakest contributions. (B) PCA biplot: PCA was applied to the full feature set to visualize marker distribution along the first two PCs (PC1 and PC2). The biplot depicts how the investigated markers cluster in distinct regions, with CRP, AFP, and thrombocytes forming separate groups from CA19-9 and EV subpopulations. AFP, alpha-fetoprotein; ALP, alkaline phosphatase; CRP, C-reactive protein; EV, extracellular vesicle; PC, principal component.

    Journal: JHEP Reports

    Article Title: AI-guided additive scoring model for differential diagnosis of primary liver cancer

    doi: 10.1016/j.jhepr.2026.101826

    Figure Lengend Snippet: Selection of the final marker set using LASSO and PCA. (A) The LASSO path plot illustrates the selection process of candidate markers using LASSO regression first. Early entrants, such as ALP, CRP, and CD9 + CD133/2 + small EVs, achieved high coefficient values, indicating consistent relevance within the fitted model. By contrast, thrombocytes, AFP, and albumin contributed with lower coefficients, whereas late entrants, including CA19-9 and CD63 + CD133/2 + small EVs, had the weakest contributions. (B) PCA biplot: PCA was applied to the full feature set to visualize marker distribution along the first two PCs (PC1 and PC2). The biplot depicts how the investigated markers cluster in distinct regions, with CRP, AFP, and thrombocytes forming separate groups from CA19-9 and EV subpopulations. AFP, alpha-fetoprotein; ALP, alkaline phosphatase; CRP, C-reactive protein; EV, extracellular vesicle; PC, principal component.

    Article Snippet: Anti-CD133/2 antibodies (130-112-195, Miltenyi Biotec) and anti-CD44v6 antibodies (130-111-238, Miltenyi Biotec) were added and titrated against their matching isotype (REA293 phycoerythrin, 130-107-771 and REA293 allophycocyanin, 130-113-446, Miltenyi Biotec) and used in concentrations according to the respective antibodies; 7-AAD (BD Pharmingen, NJ, USA, 559925) was used for dead cell exclusion.

    Techniques: Selection, Marker

    Kaplan–Meier OS analysis in LR-M patients stratified by CD133/2 small EV subpopulations. Kaplan–Meier curves depict OS (days since diagnosis) stratified by above-median versus ≤ median marker levels within disease-specific subgroups (HCC and iCCA; each n = 25). ‘Event’ indicates death. All survival analyses are exploratory and tests were two sided. (A) CD9 + CD133/2 + small EVs. Patients with iCCA (n = 25; deaths = 23; censored = 2) were stratified into above-median (n = 13) versus ≤ median (n = 12). Patients with above-median CD9 + CD133/2 + EV levels showed shorter median OS (91.0 vs . 389.5 days; median ratio: 4.28, 95% CI: 1.877–9.761). Survival differed significantly by log-rank (Mantel–Cox) test (χ 2 = 7.829, p = 0.005) and by Gehan–Breslow–Wilcoxon test (χ 2 = 5.324, p = 0.020). The log-rank HR was 2.802 (95% CI:1.164–6.744) for above-median versus ≤ median groups. Patients with HCC (n = 25; deaths = 20; censored = 5) were stratified into above-median (n = 10) versus ≤ median (n = 15). Median OS was 760.0 vs . 907.0 days. No significant survival differences were observed (log-rank χ 2 = 0.119, p = 0.730; Gehan–Breslow χ 2 = 0.0077, p = 0.930). The log-rank HR was 1.162 (95% CI: 0.482–2.798). (B) CD81 + CD133/2 + small EVs. Patients with iCCA patients (n = 25; deaths = 23; censored = 2) were stratified into above-median (n = 12) versus ≤ median (n = 13). Median OS was 114.5 vs . 173.0 days. Differences did not reach statistical significance (log-rank χ 2 = 2.840, p = 0.090; Gehan–Breslow χ 2 = 1.205, p = 0.270). The log-rank HR was 1.914 (95% CI: 0.818–4.476). Patients with HCC (n = 25; deaths = 20; censored = 5) were stratified into above-median (n = 12) versus ≤ median (n = 13). Median OS was 730.0 vs . 1460.0 days. Survival differed by log-rank test (χ 2 = 4.488, p = 0.034), whereas the Gehan–Breslow–Wilcoxon test did not reach significance (χ 2 = 3.048, p = 0.0808). The log-rank HR was 2.457 (95% CI: 0.996–6.064). Vertical dotted lines indicate the estimated time points at which survival reached 50% (median OS). EV, extracellular vesicle; HCC, hepatocellular carcinoma; HR, hazard ratio; iCCA, intrahepatic cholangiocarcinoma; OS, overall survival.

    Journal: JHEP Reports

    Article Title: AI-guided additive scoring model for differential diagnosis of primary liver cancer

    doi: 10.1016/j.jhepr.2026.101826

    Figure Lengend Snippet: Kaplan–Meier OS analysis in LR-M patients stratified by CD133/2 small EV subpopulations. Kaplan–Meier curves depict OS (days since diagnosis) stratified by above-median versus ≤ median marker levels within disease-specific subgroups (HCC and iCCA; each n = 25). ‘Event’ indicates death. All survival analyses are exploratory and tests were two sided. (A) CD9 + CD133/2 + small EVs. Patients with iCCA (n = 25; deaths = 23; censored = 2) were stratified into above-median (n = 13) versus ≤ median (n = 12). Patients with above-median CD9 + CD133/2 + EV levels showed shorter median OS (91.0 vs . 389.5 days; median ratio: 4.28, 95% CI: 1.877–9.761). Survival differed significantly by log-rank (Mantel–Cox) test (χ 2 = 7.829, p = 0.005) and by Gehan–Breslow–Wilcoxon test (χ 2 = 5.324, p = 0.020). The log-rank HR was 2.802 (95% CI:1.164–6.744) for above-median versus ≤ median groups. Patients with HCC (n = 25; deaths = 20; censored = 5) were stratified into above-median (n = 10) versus ≤ median (n = 15). Median OS was 760.0 vs . 907.0 days. No significant survival differences were observed (log-rank χ 2 = 0.119, p = 0.730; Gehan–Breslow χ 2 = 0.0077, p = 0.930). The log-rank HR was 1.162 (95% CI: 0.482–2.798). (B) CD81 + CD133/2 + small EVs. Patients with iCCA patients (n = 25; deaths = 23; censored = 2) were stratified into above-median (n = 12) versus ≤ median (n = 13). Median OS was 114.5 vs . 173.0 days. Differences did not reach statistical significance (log-rank χ 2 = 2.840, p = 0.090; Gehan–Breslow χ 2 = 1.205, p = 0.270). The log-rank HR was 1.914 (95% CI: 0.818–4.476). Patients with HCC (n = 25; deaths = 20; censored = 5) were stratified into above-median (n = 12) versus ≤ median (n = 13). Median OS was 730.0 vs . 1460.0 days. Survival differed by log-rank test (χ 2 = 4.488, p = 0.034), whereas the Gehan–Breslow–Wilcoxon test did not reach significance (χ 2 = 3.048, p = 0.0808). The log-rank HR was 2.457 (95% CI: 0.996–6.064). Vertical dotted lines indicate the estimated time points at which survival reached 50% (median OS). EV, extracellular vesicle; HCC, hepatocellular carcinoma; HR, hazard ratio; iCCA, intrahepatic cholangiocarcinoma; OS, overall survival.

    Article Snippet: Anti-CD133/2 antibodies (130-112-195, Miltenyi Biotec) and anti-CD44v6 antibodies (130-111-238, Miltenyi Biotec) were added and titrated against their matching isotype (REA293 phycoerythrin, 130-107-771 and REA293 allophycocyanin, 130-113-446, Miltenyi Biotec) and used in concentrations according to the respective antibodies; 7-AAD (BD Pharmingen, NJ, USA, 559925) was used for dead cell exclusion.

    Techniques: Biomarker Discovery, Marker

    Positive and negative control cells for both marker panels. ( A ) HeLa (white arrows): in the first panel positive for cytokeratin (CK), Vimentin (Vim), VEGF and in the second panel positive for p16INK4A, negative for PD-L1 and CD45. ( B ) T98G (red arrows): positive for Vim. ( C ) CaSki (yellow arrows): positive for CK, Vim, VEGF, PD-L1 and p16INK4A. ( D ) MCF-7 (green arrows): positive for CK. All cell lines were negative for CD45. Only hematopoietic cells showed CD45 positive staining. Scale bar = 10 μm. Images were processed using Zeiss ZEN 3.7 software and the figure was assembled using Microsoft PowerPoint.

    Journal: International Journal of Molecular Sciences

    Article Title: Disseminated Tumor Cells (DTCs) in Patients with Cervical Cancer Reveal Mesenchymal Properties and Potential Therapeutic Targets—A New Perspective?

    doi: 10.3390/ijms27114875

    Figure Lengend Snippet: Positive and negative control cells for both marker panels. ( A ) HeLa (white arrows): in the first panel positive for cytokeratin (CK), Vimentin (Vim), VEGF and in the second panel positive for p16INK4A, negative for PD-L1 and CD45. ( B ) T98G (red arrows): positive for Vim. ( C ) CaSki (yellow arrows): positive for CK, Vim, VEGF, PD-L1 and p16INK4A. ( D ) MCF-7 (green arrows): positive for CK. All cell lines were negative for CD45. Only hematopoietic cells showed CD45 positive staining. Scale bar = 10 μm. Images were processed using Zeiss ZEN 3.7 software and the figure was assembled using Microsoft PowerPoint.

    Article Snippet: In the first round of staining, anti-human Pan-Cytokeratin APC-conjugated REAdye_lease antibody (Cat. No. 130-123-091, Miltenyi Biotech, Bergisch Gladbach, Germany), anti-human vimentin FITC-conjugated REAdye_lease antibody (Cat. No. 130-127-022, Miltenyi Biotech, Bergisch Gladbach, Germany) and anti-human VEGF PE-conjugated REAdye_lease antibody (Cat. No. 130-118-061, Miltenyi Biotech, Bergisch Gladbach, Germany) were applied.

    Techniques: Negative Control, Marker, Staining, Software

    Representative images of patient-derived DTCs with three different profiles. ( A ) DTC positive for Vim, VEGF and p16, negative for CK, PD-L1 and CD45. ( B ) CK and Vim positive while negative for VEGF, PD-L1, p16 and CD45. ( C ) DTC positive for Vim and PD-L1, negative for all other markers. Scale bar = 10 μm. Images were processed using Zeiss ZEN 3.7 software and figure was assembled using Microsoft PowerPoint.

    Journal: International Journal of Molecular Sciences

    Article Title: Disseminated Tumor Cells (DTCs) in Patients with Cervical Cancer Reveal Mesenchymal Properties and Potential Therapeutic Targets—A New Perspective?

    doi: 10.3390/ijms27114875

    Figure Lengend Snippet: Representative images of patient-derived DTCs with three different profiles. ( A ) DTC positive for Vim, VEGF and p16, negative for CK, PD-L1 and CD45. ( B ) CK and Vim positive while negative for VEGF, PD-L1, p16 and CD45. ( C ) DTC positive for Vim and PD-L1, negative for all other markers. Scale bar = 10 μm. Images were processed using Zeiss ZEN 3.7 software and figure was assembled using Microsoft PowerPoint.

    Article Snippet: In the first round of staining, anti-human Pan-Cytokeratin APC-conjugated REAdye_lease antibody (Cat. No. 130-123-091, Miltenyi Biotech, Bergisch Gladbach, Germany), anti-human vimentin FITC-conjugated REAdye_lease antibody (Cat. No. 130-127-022, Miltenyi Biotech, Bergisch Gladbach, Germany) and anti-human VEGF PE-conjugated REAdye_lease antibody (Cat. No. 130-118-061, Miltenyi Biotech, Bergisch Gladbach, Germany) were applied.

    Techniques: Derivative Assay, Software

    Comparison of p16, VEGF and PD-L1 in matching tumor tissue. In four cases we found VEGF on DTCs but not in matching tumor tissue, indicating a potential therapeutic target. Further, we detected PD-L1 on DTCs in 11 patients that had PD-L1 negative tissue at diagnosis, suggesting a potential discordance between tumor tissue and DTC phenotype. Chart generated using Microsoft Excel.

    Journal: International Journal of Molecular Sciences

    Article Title: Disseminated Tumor Cells (DTCs) in Patients with Cervical Cancer Reveal Mesenchymal Properties and Potential Therapeutic Targets—A New Perspective?

    doi: 10.3390/ijms27114875

    Figure Lengend Snippet: Comparison of p16, VEGF and PD-L1 in matching tumor tissue. In four cases we found VEGF on DTCs but not in matching tumor tissue, indicating a potential therapeutic target. Further, we detected PD-L1 on DTCs in 11 patients that had PD-L1 negative tissue at diagnosis, suggesting a potential discordance between tumor tissue and DTC phenotype. Chart generated using Microsoft Excel.

    Article Snippet: In the first round of staining, anti-human Pan-Cytokeratin APC-conjugated REAdye_lease antibody (Cat. No. 130-123-091, Miltenyi Biotech, Bergisch Gladbach, Germany), anti-human vimentin FITC-conjugated REAdye_lease antibody (Cat. No. 130-127-022, Miltenyi Biotech, Bergisch Gladbach, Germany) and anti-human VEGF PE-conjugated REAdye_lease antibody (Cat. No. 130-118-061, Miltenyi Biotech, Bergisch Gladbach, Germany) were applied.

    Techniques: Comparison, Biomarker Discovery, Generated

    Study design. At diagnosis a tissue biopsy was obtained to histologically confirm cervical cancer cases. The FFPE tissue samples were stained against p16, PD-L1 and VEGF for comparison with DTC profiles. One day prior to oncologic surgery, blood samples were collected for CTC detection using the standardized CK based brightfield method. Bone marrow aspirates were sampled during surgery and DTCs were analyzed using the CK based and the multi-parameter IF staining method. Post-operative follow-up blood samples were collected within two years after surgery during routine visits if applicable. Clinical Outcome was assessed up to February 2025. Created with BioRender.com.

    Journal: International Journal of Molecular Sciences

    Article Title: Disseminated Tumor Cells (DTCs) in Patients with Cervical Cancer Reveal Mesenchymal Properties and Potential Therapeutic Targets—A New Perspective?

    doi: 10.3390/ijms27114875

    Figure Lengend Snippet: Study design. At diagnosis a tissue biopsy was obtained to histologically confirm cervical cancer cases. The FFPE tissue samples were stained against p16, PD-L1 and VEGF for comparison with DTC profiles. One day prior to oncologic surgery, blood samples were collected for CTC detection using the standardized CK based brightfield method. Bone marrow aspirates were sampled during surgery and DTCs were analyzed using the CK based and the multi-parameter IF staining method. Post-operative follow-up blood samples were collected within two years after surgery during routine visits if applicable. Clinical Outcome was assessed up to February 2025. Created with BioRender.com.

    Article Snippet: In the first round of staining, anti-human Pan-Cytokeratin APC-conjugated REAdye_lease antibody (Cat. No. 130-123-091, Miltenyi Biotech, Bergisch Gladbach, Germany), anti-human vimentin FITC-conjugated REAdye_lease antibody (Cat. No. 130-127-022, Miltenyi Biotech, Bergisch Gladbach, Germany) and anti-human VEGF PE-conjugated REAdye_lease antibody (Cat. No. 130-118-061, Miltenyi Biotech, Bergisch Gladbach, Germany) were applied.

    Techniques: Biomarker Discovery, Staining, Comparison

    Sequential multi-parameter immunofluorescent staining. After applying the first antibody panel (CK, Vim and VEGF), the slides were scanned and DTC profiles were assessed. Subsequently, releasable fluorochrome-conjugates were digested and the slides were subjected to the second antibody panel (PD-L1, p16, CD45) followed by scanning and DTC detection. Created with BioRender.com.

    Journal: International Journal of Molecular Sciences

    Article Title: Disseminated Tumor Cells (DTCs) in Patients with Cervical Cancer Reveal Mesenchymal Properties and Potential Therapeutic Targets—A New Perspective?

    doi: 10.3390/ijms27114875

    Figure Lengend Snippet: Sequential multi-parameter immunofluorescent staining. After applying the first antibody panel (CK, Vim and VEGF), the slides were scanned and DTC profiles were assessed. Subsequently, releasable fluorochrome-conjugates were digested and the slides were subjected to the second antibody panel (PD-L1, p16, CD45) followed by scanning and DTC detection. Created with BioRender.com.

    Article Snippet: In the first round of staining, anti-human Pan-Cytokeratin APC-conjugated REAdye_lease antibody (Cat. No. 130-123-091, Miltenyi Biotech, Bergisch Gladbach, Germany), anti-human vimentin FITC-conjugated REAdye_lease antibody (Cat. No. 130-127-022, Miltenyi Biotech, Bergisch Gladbach, Germany) and anti-human VEGF PE-conjugated REAdye_lease antibody (Cat. No. 130-118-061, Miltenyi Biotech, Bergisch Gladbach, Germany) were applied.

    Techniques: Staining

    Box plot showing the residual fluorescence signal after the release step (%) in 295 CaSki cells following treatment with a VEGF-PE conjugated antibody. The release efficiency was evaluated by signal quantification in the cells before and after treatment with the release reagent. The median fluorescence signal was 24.5% (mean 26.7%), corresponding to a signal reduction of 75.5%. The range was 1.75% to 87.78%. Statistical graph was generated using IBM SPSS Statistics Version 29.0.

    Journal: International Journal of Molecular Sciences

    Article Title: Disseminated Tumor Cells (DTCs) in Patients with Cervical Cancer Reveal Mesenchymal Properties and Potential Therapeutic Targets—A New Perspective?

    doi: 10.3390/ijms27114875

    Figure Lengend Snippet: Box plot showing the residual fluorescence signal after the release step (%) in 295 CaSki cells following treatment with a VEGF-PE conjugated antibody. The release efficiency was evaluated by signal quantification in the cells before and after treatment with the release reagent. The median fluorescence signal was 24.5% (mean 26.7%), corresponding to a signal reduction of 75.5%. The range was 1.75% to 87.78%. Statistical graph was generated using IBM SPSS Statistics Version 29.0.

    Article Snippet: In the first round of staining, anti-human Pan-Cytokeratin APC-conjugated REAdye_lease antibody (Cat. No. 130-123-091, Miltenyi Biotech, Bergisch Gladbach, Germany), anti-human vimentin FITC-conjugated REAdye_lease antibody (Cat. No. 130-127-022, Miltenyi Biotech, Bergisch Gladbach, Germany) and anti-human VEGF PE-conjugated REAdye_lease antibody (Cat. No. 130-118-061, Miltenyi Biotech, Bergisch Gladbach, Germany) were applied.

    Techniques: Fluorescence, Generated

    CaSki cells before and after the release step. Representative immunofluorescence image of two CaSki cells spiked into bone marrow cells ( A ) before and ( B ) after the release step. The PE channel shows VEGF staining before staining with CD45, while nuclei were stained with DAPI (blue). Images were processed using Zeiss ZEN software and figure was assembled using Microsoft PowerPoint.

    Journal: International Journal of Molecular Sciences

    Article Title: Disseminated Tumor Cells (DTCs) in Patients with Cervical Cancer Reveal Mesenchymal Properties and Potential Therapeutic Targets—A New Perspective?

    doi: 10.3390/ijms27114875

    Figure Lengend Snippet: CaSki cells before and after the release step. Representative immunofluorescence image of two CaSki cells spiked into bone marrow cells ( A ) before and ( B ) after the release step. The PE channel shows VEGF staining before staining with CD45, while nuclei were stained with DAPI (blue). Images were processed using Zeiss ZEN software and figure was assembled using Microsoft PowerPoint.

    Article Snippet: In the first round of staining, anti-human Pan-Cytokeratin APC-conjugated REAdye_lease antibody (Cat. No. 130-123-091, Miltenyi Biotech, Bergisch Gladbach, Germany), anti-human vimentin FITC-conjugated REAdye_lease antibody (Cat. No. 130-127-022, Miltenyi Biotech, Bergisch Gladbach, Germany) and anti-human VEGF PE-conjugated REAdye_lease antibody (Cat. No. 130-118-061, Miltenyi Biotech, Bergisch Gladbach, Germany) were applied.

    Techniques: Immunofluorescence, Staining, Software

    Effect of MIT-A on stemness-associated markers in HCC cells. ( A ) Heatmap showing the expression of stemness-associated genes ( PROM1 /CD133, CD24 , MYC , ITGB1 , CD44 , EPCAM , KLF4 ) and direct SP1 targets ( SETDB1 , DNMT3A , DNMT3B ) in Huh7 WT and SR cells after MIT-A treatment. ( B ) RT-qPCR analysis of SP1, pluripotency markers ( NANOG , SOX2 , POU5F1 , KLF4 ), and surface markers ( CD24 , PROM1/ CD133 ) in Huh7 WT, Huh7 SR, and HepG2 cells treated with MIT-A (200 nM, 24 h for Huh7 WT and HepG2; 400 nM, 24 h for Huh7 SR). Expression was normalized to 18S. ( C ) Western blot analysis of stemness regulators (OCT4, NANOG, CD133), SP1, and SETDB1 in Huh7 WT and SR cells treated with MIT-A. β-actin served as loading control. Representative blots and quantifications are shown. ( D ) Flow cytometry analysis of CSC surface markers (CD24, CD133) in Huh7 WT, Huh7 SR, and HepG2 cells after MIT-A exposure. Representative histograms are shown for Huh7 WT cells (red, control; violet, MIT-A; blue, isotype). Mean fluorescence intensity (MFI) values are shown on the right. Quantification is presented as bar graphs. Data are presented as mean ± SEM from independent experiments (( B , C ): n = 3; ( D ): n = 4–6). Statistical analyses were performed using unpaired two-tailed Student’s t -tests ( B ) or Welch’s t -tests when appropriate ( C , D ). Statistical significance is indicated as * p < 0.05, ** p < 0.01, *** p < 0.001; NS, not significant.

    Journal: Cells

    Article Title: Pharmacological Inhibition of SP1 Reverses Cancer Stemness and Enhances Sorafenib Efficacy in Hepatocellular Carcinoma

    doi: 10.3390/cells15110961

    Figure Lengend Snippet: Effect of MIT-A on stemness-associated markers in HCC cells. ( A ) Heatmap showing the expression of stemness-associated genes ( PROM1 /CD133, CD24 , MYC , ITGB1 , CD44 , EPCAM , KLF4 ) and direct SP1 targets ( SETDB1 , DNMT3A , DNMT3B ) in Huh7 WT and SR cells after MIT-A treatment. ( B ) RT-qPCR analysis of SP1, pluripotency markers ( NANOG , SOX2 , POU5F1 , KLF4 ), and surface markers ( CD24 , PROM1/ CD133 ) in Huh7 WT, Huh7 SR, and HepG2 cells treated with MIT-A (200 nM, 24 h for Huh7 WT and HepG2; 400 nM, 24 h for Huh7 SR). Expression was normalized to 18S. ( C ) Western blot analysis of stemness regulators (OCT4, NANOG, CD133), SP1, and SETDB1 in Huh7 WT and SR cells treated with MIT-A. β-actin served as loading control. Representative blots and quantifications are shown. ( D ) Flow cytometry analysis of CSC surface markers (CD24, CD133) in Huh7 WT, Huh7 SR, and HepG2 cells after MIT-A exposure. Representative histograms are shown for Huh7 WT cells (red, control; violet, MIT-A; blue, isotype). Mean fluorescence intensity (MFI) values are shown on the right. Quantification is presented as bar graphs. Data are presented as mean ± SEM from independent experiments (( B , C ): n = 3; ( D ): n = 4–6). Statistical analyses were performed using unpaired two-tailed Student’s t -tests ( B ) or Welch’s t -tests when appropriate ( C , D ). Statistical significance is indicated as * p < 0.05, ** p < 0.01, *** p < 0.001; NS, not significant.

    Article Snippet: Membranes were probed with primary antibodies against SP1 (Santa Cruz Biotechnology, Dallas, TX, USA, #sc-420, 1/200), SETDB1 (Abcam, Cambridge, UK, #ab-107225, 1/2000), XIAP (BD Transduction Laboratories, BD Biosciences, San Jose, CA, USA, #Mab 59520, 1/500), BCL-XL (Santa Cruz Biotechnology, Dallas, TX, USA, #sc634, 1/200), BAX (NT) (Upstate, Merck Millipore, Burlington, MA, USA, #06-499, 1/250), E-cadherin (BD Biosciences, San Jose, CA, USA, #610181, 1/1000), EpCam (Bio-Techne, Minneapolis, MN, USA, #AF960, 1/1000), vimentin (Cell signaling Technology, #73260, 1/1000), SOX2 (Bio-Techne, Minneapolis, MN, USA, #MAB2018, 1/500), Oct4 (Bio-Techne, Minneapolis, MN, USA, #AF1759, 1/1000), Nanog (Santa Cruz Biotechnology, Dallas, TX, USA, #sc-134218, 1/200), CD133 (Miltenyi Biotec, Bergisch Gladbach, Germany, #130-092-395, 1/200), CD24 (Santa Cruz Biotechnology, Dallas, TX, USA, #sc11406, 1/200), GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA, #sc-47724, 1/500) and β-actin (Cell Signaling Technology, Danvers, MA, USA, #47778, 1/15,000), followed by HRP-conjugated secondary antibodies.

    Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control, Flow Cytometry, Fluorescence, Two Tailed Test

    Genetic silencing of SP1 partially recapitulates the effects of pharmacological inhibition in HCC cells. ( A ) Validation of SP1 knockdown by RT-qPCR. Huh7 cells were transiently transfected with SP1-specific siRNA (siSP1) or control siRNA, with MIT-A (200 nM) included as a pharmacological control. SP1 mRNA expression was assessed 24 h post-transfection by RT-qPCR and normalized to 18S. Data are presented as mean ± SEM from at least three independent experiments. ( B ) Validation of SP1 protein downregulation by Western blot. SP1 protein levels were analyzed 48 h post-transfection by immunoblotting. β-actin served as loading control. Representative blots and densitometric quantification from three independent experiments are shown (mean ± SEM). ( C ) Effect of SP1 silencing on cell proliferation. Forty-eight hours after siRNA transfection, cell proliferation was monitored using the CellTiter-Glo luminescent assay and followed for 24–72 h. Luminescence values were normalized to the initial time point. Data represent mean ± SEM from at least three independent experiments. ( D ) Effect of SP1 silencing on stemness-associated transcription factors. Expression of OCT4 (POU5F1), NANOG, SOX2, SETDB1, PROM1/CD133, and KLF4 was evaluated by RT-qPCR 48 h post-transfection. Expression levels were normalized to 18S. Data are presented as mean ± SEM from at least three independent experiments. ( E ) Western blot validation of OCT4 and NANOG downregulation following SP1 silencing. Representative blots and densitometric quantification from three independent experiments are shown (mean ± SEM). ( F ) Flow cytometry analysis of CSC surface markers following SP1 knockdown. The proportion of CD24 + and CD133 + cells was quantified 48 h post-transfection. ( G ) Analysis of aldehyde dehydrogenase (ALDH) activity following SP1 silencing. The percentage of ALDH-positive cells and mean fluorescence intensity (MFI) were assessed by flow cytometry. ( H , I ) Effect of SP1 silencing on sorafenib sensitivity. Forty-eight hours after siRNA transfection, Huh7 cells were exposed to increasing concentrations of sorafenib (0, 2, 5, 10, and 20 µM). ( H ) Cell viability was assessed using the CellTiter-Glo assay to determine sorafenib IC 50 values. ( I ) Viability was independently evaluated by fluorescein diacetate (FDA) staining followed by flow cytometry. Data are presented as mean ± SEM from independent experiments (( A – F , H ): n = 3; ( G ): n = 3; ( I ): n = 4). For experiments involving multiple time points or drug concentrations ( C , H , I ), statistical analyses were performed using two-way ANOVA followed by Sidak’s multiple comparisons test. For comparisons between two groups ( A , B , D – G ), unpaired two-tailed Student’s t -test was used. Statistical significance is indicated as * p < 0.05, ** p < 0.01, *** p < 0.001; NS, not significant.

    Journal: Cells

    Article Title: Pharmacological Inhibition of SP1 Reverses Cancer Stemness and Enhances Sorafenib Efficacy in Hepatocellular Carcinoma

    doi: 10.3390/cells15110961

    Figure Lengend Snippet: Genetic silencing of SP1 partially recapitulates the effects of pharmacological inhibition in HCC cells. ( A ) Validation of SP1 knockdown by RT-qPCR. Huh7 cells were transiently transfected with SP1-specific siRNA (siSP1) or control siRNA, with MIT-A (200 nM) included as a pharmacological control. SP1 mRNA expression was assessed 24 h post-transfection by RT-qPCR and normalized to 18S. Data are presented as mean ± SEM from at least three independent experiments. ( B ) Validation of SP1 protein downregulation by Western blot. SP1 protein levels were analyzed 48 h post-transfection by immunoblotting. β-actin served as loading control. Representative blots and densitometric quantification from three independent experiments are shown (mean ± SEM). ( C ) Effect of SP1 silencing on cell proliferation. Forty-eight hours after siRNA transfection, cell proliferation was monitored using the CellTiter-Glo luminescent assay and followed for 24–72 h. Luminescence values were normalized to the initial time point. Data represent mean ± SEM from at least three independent experiments. ( D ) Effect of SP1 silencing on stemness-associated transcription factors. Expression of OCT4 (POU5F1), NANOG, SOX2, SETDB1, PROM1/CD133, and KLF4 was evaluated by RT-qPCR 48 h post-transfection. Expression levels were normalized to 18S. Data are presented as mean ± SEM from at least three independent experiments. ( E ) Western blot validation of OCT4 and NANOG downregulation following SP1 silencing. Representative blots and densitometric quantification from three independent experiments are shown (mean ± SEM). ( F ) Flow cytometry analysis of CSC surface markers following SP1 knockdown. The proportion of CD24 + and CD133 + cells was quantified 48 h post-transfection. ( G ) Analysis of aldehyde dehydrogenase (ALDH) activity following SP1 silencing. The percentage of ALDH-positive cells and mean fluorescence intensity (MFI) were assessed by flow cytometry. ( H , I ) Effect of SP1 silencing on sorafenib sensitivity. Forty-eight hours after siRNA transfection, Huh7 cells were exposed to increasing concentrations of sorafenib (0, 2, 5, 10, and 20 µM). ( H ) Cell viability was assessed using the CellTiter-Glo assay to determine sorafenib IC 50 values. ( I ) Viability was independently evaluated by fluorescein diacetate (FDA) staining followed by flow cytometry. Data are presented as mean ± SEM from independent experiments (( A – F , H ): n = 3; ( G ): n = 3; ( I ): n = 4). For experiments involving multiple time points or drug concentrations ( C , H , I ), statistical analyses were performed using two-way ANOVA followed by Sidak’s multiple comparisons test. For comparisons between two groups ( A , B , D – G ), unpaired two-tailed Student’s t -test was used. Statistical significance is indicated as * p < 0.05, ** p < 0.01, *** p < 0.001; NS, not significant.

    Article Snippet: Membranes were probed with primary antibodies against SP1 (Santa Cruz Biotechnology, Dallas, TX, USA, #sc-420, 1/200), SETDB1 (Abcam, Cambridge, UK, #ab-107225, 1/2000), XIAP (BD Transduction Laboratories, BD Biosciences, San Jose, CA, USA, #Mab 59520, 1/500), BCL-XL (Santa Cruz Biotechnology, Dallas, TX, USA, #sc634, 1/200), BAX (NT) (Upstate, Merck Millipore, Burlington, MA, USA, #06-499, 1/250), E-cadherin (BD Biosciences, San Jose, CA, USA, #610181, 1/1000), EpCam (Bio-Techne, Minneapolis, MN, USA, #AF960, 1/1000), vimentin (Cell signaling Technology, #73260, 1/1000), SOX2 (Bio-Techne, Minneapolis, MN, USA, #MAB2018, 1/500), Oct4 (Bio-Techne, Minneapolis, MN, USA, #AF1759, 1/1000), Nanog (Santa Cruz Biotechnology, Dallas, TX, USA, #sc-134218, 1/200), CD133 (Miltenyi Biotec, Bergisch Gladbach, Germany, #130-092-395, 1/200), CD24 (Santa Cruz Biotechnology, Dallas, TX, USA, #sc11406, 1/200), GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA, #sc-47724, 1/500) and β-actin (Cell Signaling Technology, Danvers, MA, USA, #47778, 1/15,000), followed by HRP-conjugated secondary antibodies.

    Techniques: Inhibition, Biomarker Discovery, Knockdown, Quantitative RT-PCR, Transfection, Control, Expressing, Western Blot, Luminescence Assay, Flow Cytometry, Activity Assay, Fluorescence, Glo Assay, Staining, Two Tailed Test