modcs expressing cd11b  (Miltenyi Biotec)

Journal: iScience

Article Title: Disruption of CD47-SIRPα signaling restores inflammatory function in tumor-associated myeloid-derived suppressor cells

doi: 10.1016/j.isci.2024.109546

Figure Lengend Snippet: The myeloid compartment in B16.F10 melanoma shifts toward suppressive phenotypes as tumors develop (A) Flow cytometry quantification of the percentage of DCs (CD11c+) and phagocytes (CD11b+CD11c-) within CD45 + cells. (B) Flow cytometry quantification of MDSCs populations (moDCs; CD11b + CD11c + Ly6C + , M-MDSCs; CD11b + CD11c − Ly6C + , and G-MDSCs; CD11b + CD11c − Ly6G + ) within CD45 + cells. (C) Flow cytometry quantification of the expression levels of immune-modulatory markers PD-L1, FasL, ARG1, and NOS2 by moDCs, M-MDSCs, and G-MDSCs. (D) Representative CFSE plots for CD8 T cell proliferation after culture alone, co-culture with tumor-derived CD11b + Ly6C − cells, or co-culture with a mix of tumor-derived moDCs and M-MDSCs. Black bar highlights the gated proliferated cells. (E) Flow cytometry quantification of the percentage of proliferating CD8 and CD4 cells cultured alone, co-cultured with tumor-derived CD11b + Ly6C − cells, or a mix of moDCs and M-MDSCs. (F) Representative CFSE plots for CD4 and CD8 T cell proliferation after co-culture with pre-sorted, tumor-derived moDCs or M-MDSCs. Black bar highlights the gated proliferated cells. (G) Quantification of T cell suppression following incubation with pre-sorted, tumor-derived moDCs and M-MDSCs compared to T cells cultured alone. Data are mean ± SEM; ∗∗ = p < 0.01, ∗∗∗ = p < 0.001, ∗∗∗∗ = p < 0.0001. (A–C) Mixed effect analysis with a Tukey’s post hoc test. (E and G) One-way ANOVA with a Tukey’s post hoc test. (A and B) n = 5 mice for day-5 and day-9 tumors and n = 6 for day-11 tumors, from two independent experiments comparing each cell type at day 5, 9, or 11 time points with the day 0 time point. (C) n = 8 mice for day 5 tumors and n = 6 mice for day 11 tumors from two independent experiments. (E) n = 3 and (G) n = 2 mice performed in duplicate from two different experiments.

Article Snippet: M-MDSCs and moDCs expressing CD11b and Ly6C were harvested using two different MACs kits, one to isolate CD11b+ cells (Miltenyi Biotec Cat: 130-113-233) and subsequently Ly6C + cells (Miltenyi Biotec Cat: 130-111-776) as per the manufacturer's instructions.

Techniques: Flow Cytometry, Expressing, Co-Culture Assay, Derivative Assay, Cell Culture, Incubation

Journal: iScience

Article Title: Disruption of CD47-SIRPα signaling restores inflammatory function in tumor-associated myeloid-derived suppressor cells

doi: 10.1016/j.isci.2024.109546

Figure Lengend Snippet: Distribution of CD47 and SIRPα expression across the TME (A) Clustering of stromal populations identified in B16.F10 melanomas and matched draining lymph nodes analyzed from data previously published by Davidson et al. (B) Expression of CD47 and its cognate receptor, SIRPα, distributed across stromal clusters. (C) Violin plots highlighting widespread CD47 but restricted SIRPα expression across stromal subsets. (D) Flow cytometry quantification of CD47 expression at the protein level in T cells, (immunomodulatory) CAF 1, (myofibroblast) CAF 2, myeloid cells, endothelial cells (CD31 + ), and B16.F10 tumor cells. (E) Representative confocal image of a day 11 B16.F10 melanoma showing myeloid populations. Arrows indicate CD11b+Ly6C + SIRPα+ cells. Insets show zoom of selected ROI. Arrowheads depict cells positive for CD11b but negative for Ly6C and SIRPα. DAPI (gray), CD11b (red), Ly6C (green), SIRPα (blue). Scale bar is 50μm. Data are mean ± SEM; ∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001, ∗∗∗∗ = p < 0.0001. (D) One-way ANOVA with a Dunnett post hoc test. (D) n = 3 replicates from two independent experiments.

Article Snippet: M-MDSCs and moDCs expressing CD11b and Ly6C were harvested using two different MACs kits, one to isolate CD11b+ cells (Miltenyi Biotec Cat: 130-113-233) and subsequently Ly6C + cells (Miltenyi Biotec Cat: 130-111-776) as per the manufacturer's instructions.

Techniques: Expressing, Flow Cytometry

Journal: iScience

Article Title: Disruption of CD47-SIRPα signaling restores inflammatory function in tumor-associated myeloid-derived suppressor cells

doi: 10.1016/j.isci.2024.109546

Figure Lengend Snippet:

Article Snippet: M-MDSCs and moDCs expressing CD11b and Ly6C were harvested using two different MACs kits, one to isolate CD11b+ cells (Miltenyi Biotec Cat: 130-113-233) and subsequently Ly6C + cells (Miltenyi Biotec Cat: 130-111-776) as per the manufacturer's instructions.

Techniques: Purification, Recombinant, Modification, Red Blood Cell Lysis, Cell Isolation, Staining, ATP Assay, Quantitation Assay, Software