Journal: Journal of experimental & clinical cancer research : CR
Article Title: Combinatorial macrophage induced innate immunotherapy against Ewing sarcoma: Turning "Two Keys" simultaneously.
doi: 10.1186/s13046-024-03093-w
Figure Lengend Snippet: Fig. 5 Characterization of markers for ES-phagocytic macrophages. A Gating strategy for evaluating cell surface protein expression on phagocytic versus non-phagocytic macrophages. In-vitro phagocytosis assay was carried out incubating A673 cells (Non- or DOX- or MAG- or D&M-treated) labeled with CMFDA (detected by FITC channel) and M-CSF Mφ. Cells were harvested and stained with APC-CD11b and PE-CD38 antibodies followed by flow cytometry analysis. The viable cells were plotted in FITC (CMFDA) x APC view to visualize cell populations in the in-vitro phagocytosis assay. A gate was drawn to include cells that were FITC positive and APC positive as the phagocytic macrophage population, and another gate was drawn to include cells that were FITC- APC + as the non-phagocytic macrophage population. The FITC + APC + and FITC- APC + cells were then plotted separately in a PE x APC view to visualize expression of CD38 in both cell populations. A quat gate was drawn to visualize APC + PE + as the CD38 + and APC + PE- as the CD38- macrophages. B Comparison of percent of CD14, CD38, CD163, CD206 or CD209 positive and negative cells in the phagocytic and non-phagocytic macrophages incubated with non- or DOX- or MAG- or D&M-treated A673 cells. Macrophages alone (no A673) served as a control. *p < 0.05, **p < 0.01, ***p < 0.001 (Fisher’s Exact test). Shown are results in a representative experiment. The same trend was seen in three independent biological repeats
Article Snippet: Antibodies used include α-CD47 (B6H12, ThermoFisher Scientific, 11–0479-42), α-Calreticulin (ThermoFisher Scientific, PA3-900), α-CD11b (Miltenyi Biotec, 130–110-554), α-CD14 (R&D Systems, FAB3832P-025), α-CD38 (R&D Systems, FAB2404P), α-CD68 (BioLegend, 333,819), α-CD80 (BioLegend, 305,220), α-CD163 (R&D Systems, FAB1607P-025), α-CD206 (R&D Systems, FAB25342P), α-CD209 (R&D Systems, FAB161P-025), Alexa Fluor 488 donkey α-rabbit IgG (Invitrogen, A21206).
Techniques: Expressing, In Vitro, Phagocytosis Assay, Labeling, Staining, Flow Cytometry, Comparison, Incubation, Control
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