Anti cd8 53 6.7 pe cy

Tumor and lymph node cells isolated from mice were stained with LIVE/DEAD Fixable Violet Dead Staining Kit (Invitrogen) and labeled with a cocktail of monoclonal antibodies conjugated with fluorochromes: Anti-CD45 (30-F11) BV 605, anti-CD3 (17A2) BV 650, anti-CD4 (RM4-5) FITC, anti-CD8 (53–6.7) APC/Fire750, anti-CD25 (PC61) PE, anti-CD44 (IM7) PE-Cy7, anti-CD62L (MEL-14) PerCP-Cy5.5 (all from BioLegend), and anti-CD49b (DX5) PE-CF594 (BD Biosciences) for lymphoid cell analysis in tumors and anti-CD4 (RM4-5) PerCP-Cy5.5, anti-CD8 (53-6.7) PE-Cy, anti-CD44 (IM7) FITC, and anti-CD62L (MEL-14) BV 605 (all from BioLegend) for lymphocyte analysis in tLNs. In the following step, the cells were fixed using eBioscience Foxp3/Transcription Factor Staining Buffer Set (Invitrogen). Subsequently, cells from tLNs were labeled with anti-Ki67 (16A8) APC (BioLegend). Analyses of lymphoid cells in tumors and tLNs were performed using LSRFortessa (BD Biosciences).

Spleen cells obtained from healthy (wild-type) C57BL/6 mice were labeled with CellTrace CFSE (carboxyfluorescein succinimidyl ester) dye (0.5 µM, 15 min, 37°C; Invitrogen) and co-cultured with transduced M-MDSCs or PMN-MDSCs sorted with FACS Aria (BD Biosciences). Cells were cultured in a ratio of 1:1 (1×10⁵ splenocytes and 1×10⁵ M-MDSCs or 1×10⁴ splenocytes and 1×10⁴ PMN-MDSCs) or 2:1 (2×10⁵ splenocytes and 1×10⁵ M-MDSCs or 2×10⁴ splenocytes and 1×10⁴ PMN-MDSCs) in the presence of concanavalin A (2 µg/ml; Sigma-Aldrich) and rh IL-2 (200 U/ml, ImmunoTools). After 3 days, the cells were labeled with monoclonal antibodies conjugated with fluorochromes: Anti-CD11b (M1/70) PerCP-Cy5.5, anti-CD4 (RM4-5) APC, anti-CD8 (53–6.7) PE-Cy (all from BioLegend). CFSE mean fluorescence intensity (MFI) in CD4⁺ and CD8⁺ cells was measured using the LSRFortessa (BD Biosciences). Concentrations of interferon-γ (IFN-γ) and IL-10 in supernatants from the above co-culture were determined using ELISA kits (eBioscience, Invitrogen and BD Biosciences, respectively).