Journal: Stem Cell Research & Therapy

Article Title: Lumican from DESC spheroids enhances odontoblastic differentiation of SCAPs

doi: 10.1186/s13287-025-04849-7

Figure Lengend Snippet: Characterization of SCAPs and DESCs. a Microscopic image of the apical buds from the lower incisors of 5-7-day-old C57BL/6 mice. b Primary mixed cell population. c Purified SCAPs exhibiting spindle-shaped morphology. d Multidirectional differentiation potential of SCAPs. Alcian Blue, Alizarin Red, and Oil Red O staining confirmed glycosaminoglycan deposition, mineralized nodule formation, and lipid droplet accumulation, indicative of chondrogenic, osteogenic, and adipogenic differentiation, respectively. e , f Flow cytometry analysis revealed CD24, CD133, and CD90 positivity, and CD45 negativity in SCAPs ( n = 3). The raw dot plots are presented in Supplementary Fig. S3. g Purified DESCs displaying cobblestone-like morphology. h Immunofluorescence imaging demonstrated positive K14 expression in DESCs (K14, green; DAPI, blue). i , j Flow cytometry analysis revealed CD29 and CD49f positivity, and CD90 negativity in DESCs ( n = 3). The raw dot plots are presented in Supplementary Fig. S3

Article Snippet: For SCAP analysis, cells were labeled with allophycocyanin (APC)-conjugated CD24 antibody (APC-65121, Proteintech, China, 1:300) as well as FITC-conjugated CD133 (11-1331-82, Thermo Fisher Scientific, USA, 1:500), CD90, and CD45 (MCD4501, Thermo Fisher Scientific, USA, 1:500) antibodies.

Techniques: Purification, Staining, Flow Cytometry, Immunofluorescence, Imaging, Expressing

Journal: International Journal of Molecular Sciences

Article Title: NK Cells Lose Their Cytotoxicity Function against Cancer Stem Cell-Rich Radiotherapy-Resistant Breast Cancer Cell Populations

doi: 10.3390/ijms22179639

Figure Lengend Snippet: The larger population of CD24 −/low /CD44 + BCSCs in RT-R-breast cancer cells shows increased proliferation, colony formation, migration and invasion abilities. ( A ) CD24 −/low /CD44 + BCSCs were isolated from RT-R-MDA-MB-231 cells as described in the Materials and Methods section, and the CD24 −/low /CD44 + BCSC populations among MDA-MB-231 and RT-R-MDA-MB-231 cells were compared to the isolated CD24 −/low /CD44 + BCSCs. The same number (1 × 10 5 cells) of MDA-MB-231, RT-R-MDA-MB-231, and CD24 −/low /CD44 + cells isolated from RT-R-MDA-MB-231 cells were labeled with both an APC-conjugated anti-CD24 antibody and a PE-conjugated anti-CD44 antibody, and the population of CD24 −/low /CD44 + cells was then quantified by flow cytometry for 5 s at the same flow rate as described in the Materials and Methods section. ( B ) Cell proliferation was measured 0, 24, 48, and 72 h after seeding using a CCK-8 assay kit as described in the Materials and Methods section. ( C ) Cells seeded in 6-well plates were cultured for approximately 2 weeks with the medium replaced with fresh complete medium every 2–3 days. Then, the colony formation ability was determined as described in the Materials and Methods section. ( D , E ) Cells were seeded on an 8 μm-pore size insert with a Matrigel-coated membrane for the migration assay ( D ) or with an endothelial cell-Matrigel-coated membrane for the invasion assay ( E ). After 20 h of incubation, the migrated ( D ) or invaded cells ( E ) were stained with DAPI and counted under a fluorescence microscope. The values are presented as the mean ± SD of five independent experiments. ** p < 0.01 compared to MDA-MB-231 cells; # p < 0.05, ## p < 0.01 compared to RT-R-MDA-MB-231 cells.

Article Snippet: MDA-MB-231 and RT-R-MDA-MB-231 cells (1.4 × 10 6 ) and isolated CD24 −/low /CD44 + cells (1.4 × 10 6 ) were stained with an APC-conjugated human anti-CD24 antibody (#FAB5247A, R&D Systems, Minneapolis, MN, USA) and a PE-conjugated human anti-CD44 antibody (#FAB3660P, R&D systems).

Techniques: Migration, Isolation, Labeling, Flow Cytometry, CCK-8 Assay, Cell Culture, Pore Size, Membrane, Invasion Assay, Incubation, Staining, Fluorescence, Microscopy

Journal: Cancers

Article Title: Carcinoma-Associated Fibroblasts Promote Growth of Sox2-Expressing Breast Cancer Cells

doi: 10.3390/cancers12113435

Figure Lengend Snippet: CD44 regulates Sox2 expression, yet Sox2 hi cells are not enriched in the CD44 hi /CD24 lo cells population. ( A , B ) Tet-regulated CD44 expression in the MCF-7/F-B5 subline and its effect on Sox2 expression as determined by Western blot ( A ) or ICC analysis ( B ). ( B , right panel) The box-plot shows the differences in the fraction of Sox2 hi cells in the tet-on and tet-off modes after one day of incubation. Sox2 hi cells were counted in randomly chosen fields of view. For each condition, 40 fields were examined (~200 cells/field). Statistical analysis was performed by using the Mann-Whitney-U-test. The asterisks indicate statistical significance ( p < 0.0001). ( C , D ) CD44 expression in AnD5, LCM-AnD5 and LCMF-AnD5 cells in the presence or absence of CAF-CM ( C ) and the effect of CD44 knock-down on Sox2 expression in LCM-AnD5 and LCMF-AnD5 cells ( D ), as determined by Western blot analysis (PM = plasma membrane extract, NE = nuclear extract, FastG = Fast green). ( E ) Scatterplots of FACS profiles of LCM-AnD5 and LCMF-AnD5 cells after treatment with PE-anti-CD44 and APC-anti-CD24 antibodies. ( F ) Sox2-specific ICC of separated LCM-AnD5 and LCMF-AnD5 cell populations as indicated (left panel). For calculating the proportion of the Sox2 hi cell fractions in the three separated cell populations (right panel), P3, P4, and P5 cell fractions were separately sprayed on three slides each. For each slide, the proportion of the Sox2 hi population was determined by counting cells in 20 randomly chosen fields (10–20 cells/field). The average proportion of the Sox2 hi population in the P3, P4, and P5 fractions were compared. Statistical analysis was done with one-way ANOVA, followed by Bonferroni correction for post-hoc analysis. No statistically significant differences were found.

Article Snippet: After adjusting the cell concentration to 10 6 cells/100 μL, 2 μL anti-CD24 APC-labeled antibody (Miltenyi Biotec, 130-112-846, Bergisch Gladbach, Germany) and 2 μL anti-CD44 PE-labeled antibody (Miltenyi Biotec, 130-113-904) were added, which was followed by incubation on ice for 10 min.

Techniques: Expressing, Western Blot, Incubation, MANN-WHITNEY, Knockdown, Clinical Proteomics, Membrane

Journal: Transplantation Direct

Article Title: Autologous Mesenchymal Stromal Cells Prevent Transfusion-elicited Sensitization and Upregulate Transitional and Regulatory B Cells

doi: 10.1097/TXD.0000000000000827

Figure Lengend Snippet: MSC therapy inhibited splenic follicular B cells and plasma cells, while upregulating transitional and regulatory B cells. We examined key B cell phenotypes in the spleen and bone marrow on day 28. We noted that follicular B cells (CD45R+IgM-IgD+CD38+) and plasma cells (IgM-CD45R-CD138+) were significantly reduced in the spleen, while transitional B cells (IgD+CD45R+CD24+CD38+) and Breg cell (CD24+CD27+CD45RA+IL10+) were upregulated (A). These changes were not significant in the bone marrow, except for transitional B cells (B).

Article Snippet: For B-cell (except regulatory B [Breg] cell) stains, the antibodies used were CD45R PE-Cy7 (clone RA3-6B2; eBioscience), IgM FITC (clone G53-238, BD bioscience), IgD biotinylated (MCA190B; Bio-Rad, Hercules, CA) with Streptavidin PE-CF594 (562284, BD bioscience), CD24 APC (clone REA405; Miltenyi Biotec), CD27 BV605 (clone LG.3A10; BD Bioscience), CD38 PE (clone 14.27; BioLegend), CD138 PerCP (clone B-A38; Abcam, Cambridge, MA) and a live/dead discriminator (Ghost Dye 780; Tonbo bioscience, San Diego, CA).

Techniques: Clinical Proteomics

Journal: Cancer Cell International

Article Title: Sporadic PCDH18 somatic mutations in EpCAM-positive hepatocellular carcinoma

doi: 10.1186/s12935-017-0467-x

Figure Lengend Snippet: Hepatic stem cell marker expression in HCC1 and HCC2 cells. a Flow cytometry of HCC1 and HCC2 cells using fluorescently-labeled antibodies against EpCAM, CD133, CD90, CD44, CD24, and CD13. b Flow cytometry of EpCAM + cells using an anti-EpCAM antibody. Figure shows EpCAM + HCC1 cells on days 1, 3, and 7 after cell sorting and EpCAM + HCC2 cells on days 1, 7, and 14 after cell sorting. c Histological analysis of EpCAM + HCC1 and HCC2 xenografts. The figure shows hematoxylin and eosin (H&E) staining and anti-EpCAM immunohistochemistry (IHC) staining of the tumors. d Representative phase-contrast images of sorted EpCAM + and EpCAM − HCC1 and HCC2 cell spheroids. e EpCAM + and EpCAM − HCC1 and HCC2 spheroid formation. Experiments were performed in triplicate. Bars indicate the mean ± standard deviation. f Tumorigenic potential of EpCAM + cells. Representative photomicrographs of NOD/SCID mice (upper panel) and subcutaneous tumors (lower panel) from EpCAM + and EpCAM − HCC1 and HCC2 cell xenografts

Article Snippet: The antibodies used were: a FITC-conjugated anti-EpCAM monoclonal antibody (Clone Ber-EP4; DAKO, Carpinteria, CA); an APC-conjugated anti-CD326 (EpCAM) antibody (Miltenyi Biotec K.K., Tokyo, Japan); an APC-conjugated anti-CD90 monoclonal antibody (Clone 5E10; eBioscience, San Diego, CA); an APC-conjugated anti-CD133/2 antibody (Clone 293C3; Miltenyi Biotec K.K.); an APC-conjugated anti-CD44 mouse monoclonal antibody (eBioscience); an APC-conjugated anti-CD13 antibody (eBioscience); and a PE-conjugated anti-CD24 antibody (Miltenyi Biotec K.K.).

Techniques: Marker, Expressing, Flow Cytometry, Labeling, FACS, Staining, Immunohistochemistry, Standard Deviation

Journal: PLoS ONE

Article Title: Highly Efficient Elimination of Colorectal Tumor-Initiating Cells by an EpCAM/CD3-Bispecific Antibody Engaging Human T Cells

doi: 10.1371/journal.pone.0013474

Figure Lengend Snippet: Tumorigenicity of HT-29 colorectal cancer cell populations and primary colorectal TICs.

Article Snippet: Cells were trypsinized, washed with PBS containing 2% FCS and stained with 5–10 μg/ml of the following antibodies for 30 min at 4°C: CD44-PE (Chemicon), CD24-APC (Immunostep), CD44v6-FITC (Abcam), CD133/2-PE (Miltenyi), CD166-ALEXA 647 (Serotec), ABCG2-PE (eBioscience), E-Cadherin (SantaCruz) and EpCAM (MT110, Micromet AG).

Techniques:

Journal: PLoS ONE

Article Title: Highly Efficient Elimination of Colorectal Tumor-Initiating Cells by an EpCAM/CD3-Bispecific Antibody Engaging Human T Cells

doi: 10.1371/journal.pone.0013474

Figure Lengend Snippet: (A) Scheme of cytotoxicity assay with soft agar-based assay readout. (B) Photographs of soft agar plates with crystal violet-stained colonies growing from CD44 high /CD24 high /EpCAM high cells treated in the presence of T cells at the indicated concentrations of MT110, control BiTE antibodies at 100 ng/ml, or T cells alone. (C) Effect of MT110 on the number of CFUs per cm 2 . Bar graphs are mean values after 14 days from triplicate determinations +/− SD. Unstimulated PBMC were used as effector T cell source at an PBMC to HT-29 cell ratio of 10∶1.

Article Snippet: Cells were trypsinized, washed with PBS containing 2% FCS and stained with 5–10 μg/ml of the following antibodies for 30 min at 4°C: CD44-PE (Chemicon), CD24-APC (Immunostep), CD44v6-FITC (Abcam), CD133/2-PE (Miltenyi), CD166-ALEXA 647 (Serotec), ABCG2-PE (eBioscience), E-Cadherin (SantaCruz) and EpCAM (MT110, Micromet AG).

Techniques: Cytotoxicity Assay, Soft Agar Assay, Staining, Control

Journal: PLoS ONE

Article Title: Highly Efficient Elimination of Colorectal Tumor-Initiating Cells by an EpCAM/CD3-Bispecific Antibody Engaging Human T Cells

doi: 10.1371/journal.pone.0013474

Figure Lengend Snippet: (A) Mixtures of 5×10 5 TICs and 1×10 6 human PBMCs were inoculated into NOD/SCID mice (N = 3 for vehicle; N = 5 for vehicle + PBMCs; N = 4 for MT110 5 µg/kg; N = 3 for MT110 50 µg/kg and N = 3 for MT110 500 µg/kg group) and mice daily treated i.v. for 12 days with the indicated doses of MT110, or with vehicle controls in the absence and presence of PBMCs. The mean tumor volume is given. P-value was determined by Student's t-test. (B) Mixtures of 50,000 highly tumorigenic HT-29 xenograft-derived CD44 high /CD24 high /EpCAM high cells and 1×10 5 human PBMCs were inoculated into 5 NOD/SCID mice per group and mice daily treated i.v. for 12 days with the indicated doses of MT110, or with vehicle controls in the presence of PBMCs. The mean tumor volume is given. P-value was determined by Student's t-test. (C) Elimination of established tumors in NOD/SCID mice by treatment with MT110. Mixtures of 5×10 6 TICs and 1×10 7 human PBMCs were inoculated into 5 NOD/SCID mice per group to allow solid tumor formation. After tumor establishment at day 4 mice were treated i.v. for 14 days with 2.5 mg/kg of MT110, or with vehicle control in presence of PBMCs. The mean tumor volume is given. P-value was determined by Student's t-test.

Article Snippet: Cells were trypsinized, washed with PBS containing 2% FCS and stained with 5–10 μg/ml of the following antibodies for 30 min at 4°C: CD44-PE (Chemicon), CD24-APC (Immunostep), CD44v6-FITC (Abcam), CD133/2-PE (Miltenyi), CD166-ALEXA 647 (Serotec), ABCG2-PE (eBioscience), E-Cadherin (SantaCruz) and EpCAM (MT110, Micromet AG).

Techniques: Derivative Assay, Control