Journal: bioRxiv

Article Title: Kasugamycin inhibits melanoma lung metastasis and regulates CHI3L1-driven M2-like tumor-associated macrophage differentiation

doi: 10.1101/2025.05.26.654629

Figure Lengend Snippet: (A) Co-immunostaining of CD206 (red) and CD163 (green) in lungs challenged with B16-F10 (B16) melanoma cells with vehicle (PBS) or KSM treatment (100 mg/kg/mouse). Arrows indicate CD206/CD163 double (+) cells (yellow) or CD206 or CD163 single positive cells (white). (B) Representative FACS evaluations on CD206(+)/CD163(+), CD206(+)/ PD-L1(+), and CD163(+)/PD-L1(+) macrophages in lungs with PBS or melanoma cell challenge and KSM treatment. Scale bar in Panels A = 25 µm

Article Snippet: Protein lysates (25 μg) from lung tissues or cells were subjected to SDS-PAGE, transferred to membranes, and immunoblotted with primary antibodies against CD163 (#PA5-109327, Thermo Fisher Scientific), CD206 (E6T5J, #24595S, Cell Signaling Technology, Danvers, MA, USA), phosphorylated AKT (pAkt) (193H12, #4058S, Cell Signaling Technology), and total Akt (11E7, #4685S, Cell Signaling Technology), EGFR (D38B1, #4267S, Cell Signaling Technology), phosphorylated Erk (pErk) (#9101S, Cell Signaling Technology), total Erk (#9102S, Cell Signaling Technology), and β-actin (C4, #sc-47778 HRP, Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Immunostaining

Journal: bioRxiv

Article Title: Kasugamycin inhibits melanoma lung metastasis and regulates CHI3L1-driven M2-like tumor-associated macrophage differentiation

doi: 10.1101/2025.05.26.654629

Figure Lengend Snippet: (A). Representative photographs of WT and CHI3L1 transgenic lungs with and without KSM treatment after B16-F10 melanoma cell challenge. (B) Representative H&E histology of WT and Transgenic lungs with and without KSM treatment after B16-F10 melanoma cell challenge (x4 original magnification). (C-D) The number of CD206(+)/CD163(+) and CD206(+)/PD-L1(+) macrophages detected by immunohistochemical staining in lungs of WT and CHI3L1 Tg mice with and without KSM treatment. The number of macrophages co-expressing CD206 and CD163 or PD-L1 was counted using microscopic images under 20x magnification (n = 8 each). The values in panels C and D are the mean ± SEM. *p < 0.05, **p < 0.01 ***p < 0.001 ****p < 0.0001 (One-Way ANOVA, multiple comparisons).

Article Snippet: Protein lysates (25 μg) from lung tissues or cells were subjected to SDS-PAGE, transferred to membranes, and immunoblotted with primary antibodies against CD163 (#PA5-109327, Thermo Fisher Scientific), CD206 (E6T5J, #24595S, Cell Signaling Technology, Danvers, MA, USA), phosphorylated AKT (pAkt) (193H12, #4058S, Cell Signaling Technology), and total Akt (11E7, #4685S, Cell Signaling Technology), EGFR (D38B1, #4267S, Cell Signaling Technology), phosphorylated Erk (pErk) (#9101S, Cell Signaling Technology), total Erk (#9102S, Cell Signaling Technology), and β-actin (C4, #sc-47778 HRP, Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Transgenic Assay, Immunohistochemical staining, Staining, Expressing

Journal: bioRxiv

Article Title: Kasugamycin inhibits melanoma lung metastasis and regulates CHI3L1-driven M2-like tumor-associated macrophage differentiation

doi: 10.1101/2025.05.26.654629

Figure Lengend Snippet: (A.) Schematic illustration of macrophage differentiation using human THP-1 cells. (B) Real-time PT-PCR evaluations on the M2 macrophage activation markers in the macrophages with and without CHI3L1 (500 ng/mL) and KSM (250 ng/mL) treatment. (C) Real-time qRT-PCR evaluations on the M2 macrophage activation markers in the macrophages cultured in the presence and absence of tumor cell conditioned medium (TCM; A549 cell supernatant), together with CHI3L1 and KSM treatment. (D) Representatives of immunoblot evaluations on the expression of CD206/CD163 in differentiated macrophages stimulated in the presence and absence of TCM, together with CHI3L1 and or KSM treatment. Right panel, densitometric quantitation on the blots of CD163 and CD206. The values in panels B, C, and D are mean ± SEM. *p < 0.05, **p < 0.01 ***p < 0.001 (One-Way ANOVA, multiple comparisons).

Article Snippet: Protein lysates (25 μg) from lung tissues or cells were subjected to SDS-PAGE, transferred to membranes, and immunoblotted with primary antibodies against CD163 (#PA5-109327, Thermo Fisher Scientific), CD206 (E6T5J, #24595S, Cell Signaling Technology, Danvers, MA, USA), phosphorylated AKT (pAkt) (193H12, #4058S, Cell Signaling Technology), and total Akt (11E7, #4685S, Cell Signaling Technology), EGFR (D38B1, #4267S, Cell Signaling Technology), phosphorylated Erk (pErk) (#9101S, Cell Signaling Technology), total Erk (#9102S, Cell Signaling Technology), and β-actin (C4, #sc-47778 HRP, Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Activation Assay, Quantitative RT-PCR, Cell Culture, Western Blot, Expressing, Quantitation Assay

Journal: bioRxiv

Article Title: Kasugamycin inhibits melanoma lung metastasis and regulates CHI3L1-driven M2-like tumor-associated macrophage differentiation

doi: 10.1101/2025.05.26.654629

Figure Lengend Snippet: Bulk RNAseq sequencing analysis on the differentiated macrophages from THP-1 cells was used to evaluate mRNA expression in the macrophage differentiation regulated by CHI3L1 and KSM. (A). Volcano plots showing differentially expressed genes regulated by CHI3L1. (B) Representative plots of the top 20 genes that are upregulated (> 2-fold) by CHI3L1 stimulation but downregulated (< 2-fold) by KSM treatment. (C-D) Representative mRNA and protein expression of EGFR in differentiated macrophages with CHI3L1 and KSM treatment detected by real-time qRT-PCR (left panel) and immunoblot evaluations (right panel) in the differentiated macrophages with CHI3L1 and KSM treatments. (E) Effects of EGFR inhibition on the expression of CD163 and CD206 in CHI3L1-stimulated, differentiated macrophages by gefitinib (#3000, Tocris Bioscience, Bristol, UK) treatment (0.1 and 1 μM, 72 hours). Right panel, densitometric quantitation on the blots of CD163 and CD206. The values in panels C-E are mean ± SEM. *p < 0.05, **p < 0.01 ***p < 0.001 (One-Way ANOVA, multiple comparisons).

Article Snippet: Protein lysates (25 μg) from lung tissues or cells were subjected to SDS-PAGE, transferred to membranes, and immunoblotted with primary antibodies against CD163 (#PA5-109327, Thermo Fisher Scientific), CD206 (E6T5J, #24595S, Cell Signaling Technology, Danvers, MA, USA), phosphorylated AKT (pAkt) (193H12, #4058S, Cell Signaling Technology), and total Akt (11E7, #4685S, Cell Signaling Technology), EGFR (D38B1, #4267S, Cell Signaling Technology), phosphorylated Erk (pErk) (#9101S, Cell Signaling Technology), total Erk (#9102S, Cell Signaling Technology), and β-actin (C4, #sc-47778 HRP, Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Sequencing, Expressing, Quantitative RT-PCR, Western Blot, Inhibition, Quantitation Assay

Journal: Frontiers in Immunology

Article Title: Prevalence of hybrid TLR4 + M2 monocytes/macrophages in peripheral blood and lung of systemic sclerosis patients with interstitial lung disease

doi: 10.3389/fimmu.2024.1488867

Figure Lengend Snippet: Gating strategy for the detection of circulating TLR4 + M2 and M2 monocytes in systemic sclerosis patients (SSc pts) and healthy subjects (HSs) by flow cytometry. (A) Representative dot plot of leukocyte population using CD45 and physical parameters – Side scatter (SS) and Forward scatter (INT) – after removing cell debris (B) Flow cytometry scatter dot plot to identify circulating monocytes (CD45 + CD14 + CD16 + cells) in the leukocyte population; (C) Flow cytometry scatter dot plot with quadrant regions of circulating CD163 + CD206 + cells in the monocyte population; (D) CD204 + CD163 + CD206 + cells in the monocyte population; (E) CD80 - TLR4 - CD204 + CD163 + CD206 + cells (M2 monocytes) and CD80 - TLR4 + CD204 + CD163 + CD206 + cells (TLR4 + M2 monocytes) in the CD204 + CD163 + CD206 + cells; (F, G) Flow cytometry scatter dot plot and related box plot representation of the percentage of TLR4 + M2 monocytes (CD1c - CD80 - TLR4 + CD163 + CD204 + CD206 + cells) and M2 monocytes (CD1c - CD80 - TLR4 - CD163 + CD204 + CD206 + cells) in HSs and SSc pts, excluding the presence of dendritic cells (CD1c + cells).

Article Snippet: After 1 hour in blocking solution, membranes were incubated overnight at 4°C with primary antibodies against human CD204, TLR4 (dilution 1:400 and 1:200, respectively; Santa Cruz Biotechnologies, Dallas, Texas, USA), CD206, CD163 (dilution 1:500, Cell Signaling Technology, Danvers, Massachusetts, USA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH dilution 1:1.000; Santa Cruz Biotechnologies).

Techniques: Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Prevalence of hybrid TLR4 + M2 monocytes/macrophages in peripheral blood and lung of systemic sclerosis patients with interstitial lung disease

doi: 10.3389/fimmu.2024.1488867

Figure Lengend Snippet: Immunohistochemistry of a SSc-ILD sample (SSc-83) with 40x magnification. Representative Masson’s trichrome and immunostaining for CD68, M1 markers (CD80, CD86, TLR4) and M2 markers (CD163, CD204, CD206) are depicted. Red circles highlight areas rich of collagen in Masson’s trichrome staining and areas of co-localization of TLR4 positive cells and M2 markers (CD163, CD204, CD206).

Article Snippet: After 1 hour in blocking solution, membranes were incubated overnight at 4°C with primary antibodies against human CD204, TLR4 (dilution 1:400 and 1:200, respectively; Santa Cruz Biotechnologies, Dallas, Texas, USA), CD206, CD163 (dilution 1:500, Cell Signaling Technology, Danvers, Massachusetts, USA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH dilution 1:1.000; Santa Cruz Biotechnologies).

Techniques: Immunohistochemistry, Immunostaining, Staining

Journal: Frontiers in Immunology

Article Title: Prevalence of hybrid TLR4 + M2 monocytes/macrophages in peripheral blood and lung of systemic sclerosis patients with interstitial lung disease

doi: 10.3389/fimmu.2024.1488867

Figure Lengend Snippet: Percentage of positivity of all macrophage surface markers out of total cells in SSc-ILD lung samples versus controls (CNT). Three-quarters (almost 75%) of the cells found in the SSc-ILD lung samples belong to macrophage lineage. CD68, TLR4 (M1 marker), CD163 (M2 marker), CD204 (M2 marker) and CD206 (M2 marker) positive cells were found in the same areas. CD80 (M1 marker) and CD86 (M1 marker) positive cells were significantly less expressed than other markers investigated.

Article Snippet: After 1 hour in blocking solution, membranes were incubated overnight at 4°C with primary antibodies against human CD204, TLR4 (dilution 1:400 and 1:200, respectively; Santa Cruz Biotechnologies, Dallas, Texas, USA), CD206, CD163 (dilution 1:500, Cell Signaling Technology, Danvers, Massachusetts, USA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH dilution 1:1.000; Santa Cruz Biotechnologies).

Techniques: Marker

Journal: Frontiers in Immunology

Article Title: Prevalence of hybrid TLR4 + M2 monocytes/macrophages in peripheral blood and lung of systemic sclerosis patients with interstitial lung disease

doi: 10.3389/fimmu.2024.1488867

Figure Lengend Snippet: Immunohistochemistry of a control lung sample (CNT-A6) with 40x magnification. Representative Masson’s trichrome and immunostaining for CD68, M1 markers (CD80, CD86, TLR4) and M2 markers (CD163, CD204, CD206) are depicted. M1 and M2 markers are virtually absent.

Article Snippet: After 1 hour in blocking solution, membranes were incubated overnight at 4°C with primary antibodies against human CD204, TLR4 (dilution 1:400 and 1:200, respectively; Santa Cruz Biotechnologies, Dallas, Texas, USA), CD206, CD163 (dilution 1:500, Cell Signaling Technology, Danvers, Massachusetts, USA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH dilution 1:1.000; Santa Cruz Biotechnologies).

Techniques: Immunohistochemistry, Control, Immunostaining

Journal: Frontiers in Immunology

Article Title: Prevalence of hybrid TLR4 + M2 monocytes/macrophages in peripheral blood and lung of systemic sclerosis patients with interstitial lung disease

doi: 10.3389/fimmu.2024.1488867

Figure Lengend Snippet: Significant differences observed in the distribution of percentages of circulating cells expressing the monocyte/macrophage markers between systemic sclerosis (SSc) patients with or without interstitial lung disease (ILD).

Article Snippet: After 1 hour in blocking solution, membranes were incubated overnight at 4°C with primary antibodies against human CD204, TLR4 (dilution 1:400 and 1:200, respectively; Santa Cruz Biotechnologies, Dallas, Texas, USA), CD206, CD163 (dilution 1:500, Cell Signaling Technology, Danvers, Massachusetts, USA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH dilution 1:1.000; Santa Cruz Biotechnologies).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Prevalence of hybrid TLR4 + M2 monocytes/macrophages in peripheral blood and lung of systemic sclerosis patients with interstitial lung disease

doi: 10.3389/fimmu.2024.1488867

Figure Lengend Snippet: Flow cytometry analysis of circulating monocytes in systemic sclerosis (SSc) patients. Representative flow cytometry analysis to identify M2 monocytes (CD1c - CD80 - TLR4 - CD163 + CD204 + CD206 + cells) and TLR4 + M2 monocytes (CD1c - CD80 - TLR4 + CD163 + CD204 + CD206 + cells) in the peripheral blood of healthy subjects (HSs) and SSc patients with ILD (SSc-ILD) or without interstitial lung disease (SSc no-ILD). The related box plot representation of the percentage of these cells was included.

Article Snippet: After 1 hour in blocking solution, membranes were incubated overnight at 4°C with primary antibodies against human CD204, TLR4 (dilution 1:400 and 1:200, respectively; Santa Cruz Biotechnologies, Dallas, Texas, USA), CD206, CD163 (dilution 1:500, Cell Signaling Technology, Danvers, Massachusetts, USA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH dilution 1:1.000; Santa Cruz Biotechnologies).

Techniques: Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Prevalence of hybrid TLR4 + M2 monocytes/macrophages in peripheral blood and lung of systemic sclerosis patients with interstitial lung disease

doi: 10.3389/fimmu.2024.1488867

Figure Lengend Snippet: Evaluation by western blotting (WB) and related densitometric analysis of the protein synthesis of CD206, CD163, TLR4 and CD204 in cultures of monocyte-derived macrophages (MDMs) obtained from 5 voluntary healthy subjects (HS), 5 SSc patients without ILD (SSc no-ILD), and 11 SSc patients with ILD (SSc-ILD pts). The value of protein expression of CD206, CD163, TLR4 and CD204 was normalized to that of the corresponding glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in cultured HS-MDMs, MDMs from SSc no-ILD, and MDMs from SSc-ILD. The resulting value of the protein expression of each molecule in cultured MDMs from SSc-ILD and from SSc no-ILD was compared with that obtained in cultured HS-MDMs (taken as unit value). The protein expression of each molecule obtained in cultured MDMs from SSc no-ILD and SSc-ILD represent the fold increase compared to the unit value of cultured HS-MDMs. Data are reported as median with a range of fold increase compared to HSs.

Article Snippet: After 1 hour in blocking solution, membranes were incubated overnight at 4°C with primary antibodies against human CD204, TLR4 (dilution 1:400 and 1:200, respectively; Santa Cruz Biotechnologies, Dallas, Texas, USA), CD206, CD163 (dilution 1:500, Cell Signaling Technology, Danvers, Massachusetts, USA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH dilution 1:1.000; Santa Cruz Biotechnologies).

Techniques: Western Blot, Derivative Assay, Expressing, Cell Culture

Journal: Frontiers in Immunology

Article Title: Identification of common signature genes and pathways underlying the pathogenesis association between nonalcoholic fatty liver disease and heart failure

doi: 10.3389/fimmu.2024.1424308

Figure Lengend Snippet: Genes sorted by support vector machine-recursive feature elimination (SVM-RFE) method.

Article Snippet: Immunofluorescence detection involved incubating 10 μm-thick sections with primary antibodies against CD163 (#GB11340-1-100, Servicebio) and CD80 (#GB114055-100, Servicebio), followed by incubation with FITC-conjugated anti-rabbit whole IgG and Texas Red-conjugated anti-mouse whole IgG.

Techniques: Plasmid Preparation

Journal: Frontiers in Immunology

Article Title: Identification of common signature genes and pathways underlying the pathogenesis association between nonalcoholic fatty liver disease and heart failure

doi: 10.3389/fimmu.2024.1424308

Figure Lengend Snippet: Validation and receiver operating characteristic (ROC) curve analyses of CCR1 and CD163 in heart failure (HF) and nonalcoholic fatty liver disease (NAFLD). (A–D) Expression of CCR1 and CD163 in GSE26887 (A) , GSE57345 (B) , GSE126848 (C) , and GSE89632 (D) , respectively. (E–H) ROC curve and area under the curve (AUC) of CCR1 and CD163 in GSE26887 (E) , GSE57345 (F) , GSE126848 (G) , and GSE89632 (H) , respectively. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 vs. healthy controls.

Article Snippet: Immunofluorescence detection involved incubating 10 μm-thick sections with primary antibodies against CD163 (#GB11340-1-100, Servicebio) and CD80 (#GB114055-100, Servicebio), followed by incubation with FITC-conjugated anti-rabbit whole IgG and Texas Red-conjugated anti-mouse whole IgG.

Techniques: Biomarker Discovery, Expressing

Journal: Frontiers in Immunology

Article Title: Identification of common signature genes and pathways underlying the pathogenesis association between nonalcoholic fatty liver disease and heart failure

doi: 10.3389/fimmu.2024.1424308

Figure Lengend Snippet: Gene Set Enrichment Analysis (GSEA) of CCR1 and CD163 in GSE126848 and GSE26887 datasets. (A, B) Biological processes (A) and KEGG pathways (B) found by singe-gene GSEA of CCR1 in NAFLD dataset GSE126848. (C, D) Biological processes (C) and KEGG pathways (D) found by singe-gene GSEA of CD163 in NAFLD dataset GSE126848. (E, F) Biological processes (E) and KEGG pathways (F) found by singe-gene GSEA of CCR1 in HF dataset GSE26887. (G, H) Biological processes (G) and KEGG pathways (H) found by singe-gene GSEA of CD163 in HF dataset GSE26887.

Article Snippet: Immunofluorescence detection involved incubating 10 μm-thick sections with primary antibodies against CD163 (#GB11340-1-100, Servicebio) and CD80 (#GB114055-100, Servicebio), followed by incubation with FITC-conjugated anti-rabbit whole IgG and Texas Red-conjugated anti-mouse whole IgG.

Techniques:

Journal: Frontiers in Immunology

Article Title: Identification of common signature genes and pathways underlying the pathogenesis association between nonalcoholic fatty liver disease and heart failure

doi: 10.3389/fimmu.2024.1424308

Figure Lengend Snippet: Interaction of CCR1 and CD163 with inflammatory genes and chemical drugs. (A) Regulatory network of CCR1 and CD163 and their co-expression genes constructed by the GeneMANIA database. (B, C) The correlation between CCR1 and CD163 with inflammation-related genes in the GSE126848 (B) and GSE26887 (C) datasets. (D) The interactions between CCR1 and CD163 with chemicals and proteins constructed by the STITCH database.

Article Snippet: Immunofluorescence detection involved incubating 10 μm-thick sections with primary antibodies against CD163 (#GB11340-1-100, Servicebio) and CD80 (#GB114055-100, Servicebio), followed by incubation with FITC-conjugated anti-rabbit whole IgG and Texas Red-conjugated anti-mouse whole IgG.

Techniques: Expressing, Construct

Journal: Frontiers in Immunology

Article Title: Identification of common signature genes and pathways underlying the pathogenesis association between nonalcoholic fatty liver disease and heart failure

doi: 10.3389/fimmu.2024.1424308

Figure Lengend Snippet: Validation of CCR1 and CD163 in a non-alcoholic fatty liver disease mouse model. (A) Hematoxylin&eosin (H&E) staining of liver tissues in mice with normal chow (10% of calorie from fat, NC) or high-fat diet (60% of calorie from fat, HFD) for 14 weeks. The black arrow indicates infiltrated immune cells. Scale bar = 100 μm. (B) NAFLD activity score (NAS) based on the H&E staining of liver tissues. (C) Hepatic triglyceride (TG) concentrations. (D) Serum alanine aminotransferase (ALT) levels. (E) Relative mRNA expression level of inflammatory marker genes in liver tissues. (F–I) Relative mRNA expression level of Ccr1 (F) , Cd163 (G) , Cd80 (H) , and Cd206 (I) in liver tissues. (J) Representative images under fluorescence microscopy showing CD163 staining (red) and nuclear staining (DIPA, blue) of liver tissues. Scale bar = 100 μm. (K) Representative images under fluorescence microscopy showing CD80 staining (red) and nuclear staining (DIPA, blue) of liver tissues. Scale bar = 100 μm. Mean ± S.E.M., n = 12. * P< 0.05, ** P< 0.01 vs. the NC group.

Article Snippet: Immunofluorescence detection involved incubating 10 μm-thick sections with primary antibodies against CD163 (#GB11340-1-100, Servicebio) and CD80 (#GB114055-100, Servicebio), followed by incubation with FITC-conjugated anti-rabbit whole IgG and Texas Red-conjugated anti-mouse whole IgG.

Techniques: Biomarker Discovery, Staining, Activity Assay, Expressing, Marker, Fluorescence, Microscopy

Journal: Frontiers in Immunology

Article Title: Identification of common signature genes and pathways underlying the pathogenesis association between nonalcoholic fatty liver disease and heart failure

doi: 10.3389/fimmu.2024.1424308

Figure Lengend Snippet: Validation of CCR1 and CD163 in a heart failure with reduced ejection fraction (HFpEF) mouse model. (A) Wheat germ agglutinin (WGA, green) staining of heart tissues in mice with saline (CON group) or uninephrectomy surgery followed by 0.15mg/h d-aldosterone (HFpEF group) treatment for 4 weeks. Scale bar = 100 μm. (B) Quantitative results of the left ventricular cross-sectional area based on the WGA staining of heart tissues. (C) Heart weight/body weight (HW/BW) ratio. (D–F) Relative mRNA expression level of left ventricular hypertrophy markers Anp (D) , Bnp (E) , and β-MHC (F) in heart tissues. (G) Relative mRNA expression level of inflammatory marker genes in heart tissues. (H–K) Relative mRNA expression level of Ccr1 (H) , Cd163 (I) , Cd80 (J) , and Cd206 (K) in heart tissues. (L) Representative images under fluorescence microscopy showing CD163 staining (red) and nuclear staining (DIPA, blue) of heart tissues. Scale bar = 100 μm. (M) Representative images under fluorescence microscopy showing CD80 staining (red) and nuclear staining (DIPA, blue) of heart tissues. Scale bar = 100 μm. Mean ± S.E.M., n = 6. * P< 0.05, ** P< 0.01 vs. the control group.

Article Snippet: Immunofluorescence detection involved incubating 10 μm-thick sections with primary antibodies against CD163 (#GB11340-1-100, Servicebio) and CD80 (#GB114055-100, Servicebio), followed by incubation with FITC-conjugated anti-rabbit whole IgG and Texas Red-conjugated anti-mouse whole IgG.

Techniques: Biomarker Discovery, Staining, Saline, Expressing, Marker, Fluorescence, Microscopy, Control

Journal: Cell Discovery

Article Title: Integration of 3D bioprinting and multi-algorithm machine learning identified glioma susceptibilities and microenvironment characteristics

doi: 10.1038/s41421-024-00650-7

Figure Lengend Snippet: a Percentage of CD45 + cells in patient tissue and bioprinted PDTs. b Percentage of CD45 + , CD14 + , and P2RY12 + cells in patient tissues and PDTs. c Percentage of CD31 + cells in patient tissue and bioprinted PDTs. d Immunofluorescent staining of PDTs derived from 1779-HS and 29259-HS. Green: CD45. Red: GFAP. Scale bars, 100 μm. e Immunofluorescent staining of Caspase 3 in PDTs derived from 1779-HS and 29259-HS after T-cell treatment. Scale bars, 100 μm. f PCA of drug responses in GSC, Mg, and Mo under different culture conditions. 2D-Mo: 2D cultured monocyte. G-Mo: GSC co-cultured monocyte. 2D-Mg: 2D cultured microglia. G-Mg: GSC co-cultured microglia. Sphere: traditional sphere cultured GSC. G-GSC: microglia-coculturedd GSC. O-GSC: monocyte co-cultured GSC. g Cytotoxicity evaluation of T-cell treatment for GSCs in bioprinted GBM-Mg and GBM-Mo models, with E:T ratios of 1:1. h Cytotoxicity evaluation of bevacizumab treatment (25 μg/mL) for GSCs in bioprinted GBM-Mg and GBM-Mo models. Immunofluorescence staining of immunosuppressive markers CD163 and CD206 ( i ), and tight junction marker ZO-1 and glial cell marker GFAP ( j ) in GBM-Mg and GBM-Mo models. Scale bars, 100 μm.

Article Snippet: Following PBS washing, the cells were permeabilized with 0.1% Triton X-100 for 20 min and subsequently blocked with 5% BSA for 90 min. Primary antibodies against CD163 (1:100 dilution, Proteintech, Cat# 16646-1-AP), CD206 (1:100 dilution, Abcam, Cat# 91992 S), ZO-1 (1:100 dilution, Invitrogen, Cat# 339100), Caspase 3 (1:100 dilution, Proteintech, Cat# 66470-2-lg), CD45 (1:100 dilution, Proteintech, Cat# 65109-1-lg), or GFAP (1:100 dilution, Sigma, Cat# G3893) were diluted in staining buffer (BioLegend, Cat# 420201) and incubated at 4 °C overnight.

Techniques: Staining, Derivative Assay, Cell Culture, Immunofluorescence, Marker