Pe anti mouse gr 1 clone rb6 8c5

Single-cell suspensions of tumor-burdened lungs were re-suspended in FACS buffer (PBS, 1%BSA), blocked by non-specific staining with Fc block (anti-mouse CD16/32 mAb; BD Biosciences), The following antibodies were acquired from Biolegend and used in the flow cytometry analysis: BV605 anti-mouse CD45 (clone 30-F11), PE anti-mouse CD3 (clone 17A2), PE/Cy7 anti-mouse CD4 (clone RM4-5), BV711 anti-mouse CD8α (clone 53 − 6.7), BV421 anti-mouse Ly-6 C (clone HK1.4), PE anti-mouse Gr-1 (clone RB6-8C5), PE/Cy5 anti-mouse CD11b (clone M1/70), PE/Cy7 anti-mouse Ly-6G (clone 1A8), BV711 anti-mouse IFN-γ (clone XMG1.2), BV421 anti-mouse CD8α (clone 53 − 6.7), PE/Cy5 anti-mouse CD3 (clone 17A2). Flow cytometry data were acquired on a FACS Celesta flow cytometer and data was analysed with FlowJo V10.

The cells of interest were taken up at the final concentration of 1 x 10⁶ cells/ml in PBS (Life Technologies #14190) plus 2% Fetal bovine serum (FBS, PAN Biotech #P30-1502) and seeded (200 μl/well) into a 96-well plate (Thermo Fisher Scientific #249570). The plate was centrifuged 300g for 5 min at 4 °C, the cells were washed twice, and then incubated for 30 min at 4 °C with the respective antibodies. For NUP cells and NUP-MDSC: PerCP anti-mouse/human CD11b (clone M1/70, BioLegend #101229), FITC anti-mouse Ly6C (clone HK1.4, BioLegend #128006), APC anti-mouse CD11c (clone N418, BioLegend #117310), and PE anti-mouse Gr-1 (clone RB6-8C5, BioLegend #108407). For PMN-MDSC: PE anti-mouse/human CD11b (clone M1/70, BioLegend #101208) and APC anti-human CD15 (Biolegend #323008). For human CD34+ cells, FITC anti-human CD34 (BD #555821) was used. The cells were centrifuged and washed again twice in PBS plus FBS, and then data was acquired using iQue Screener Plus (IntelliCyt). Data analysis was done using FlowJo software (https://www.flowjo.com/).