Pe cy7 anti mouse ly 6g

On day 10 of PMN mice model, lung tissues were harvested from different treatment groups and mechanically minced and digested to obtain the single-cell-suspensions as described above. The single-cell suspensions washed with PBS and resuspended were incubated with APC-antimouse-CD11b (1:250, Cat. 101211, Biolegend, USA) and PE/Cy7-antimouse-Ly6g (1:150, Cat. 127617,Biolegend, USA) antibodies in 100 μl 1% BSA for 30 min at 4 °C in dark. After centrifuged at 400 × g and washed with PBS, cell pellets were analyzed by BD Fortessa flow cytometry. The data were analyzed using FlowJo software v10.6.2.

On day 10 of PMN mice model, lung tissues were harvested from different treatment groups and digested into single cells as introduced as above. The single-cell suspensions after washing with PBS and re-suspension were incubated with APC-antimouse-CD11b (1:250, Cat. 101211, Biolegend, USA) and PE/Cy7-antimouse-Ly6g (1:150, Cat. 127617, Biolegend, USA) antibodies in 100 μl 1% BSA for 30 min at 4 °C in the dark. After centrifuged at 400 × g and washed with PBS, CD11b⁺Ly6g⁺ MDSCs were sorted from the PMN lungs of 10–12 individual mice from each group per sample by FACS (Beckman moflo Astrios EQ). Total RNA was extracted by TRIzol for cDNA preamplification using the NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina®, then analyzed using Qubit2.0 Fluorometer, Agilent 2100 bioanalyzer and qRT-PCR. Significantly enriched gene sets were defined as p values < 0.05 comparing to the control group.