Ammonium chloride potassium lysis buffer

Red cells were removed from the splenocytes collected from the different treatment groups using ammonium-chloride-potassium lysis buffer (Beyotime Biotechnology), and the cleared splenocytes were filtered with a 70-μm cell strainer. The cells were incubated with an anti-CD16/CD32 antibody (cat. #553142; BD Biosciences, Franklin Lakes, NJ, USA) at 4°C for 15 min for Fc block. Cell viability was determined using the LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (cat. #L34957; Invitrogen). Then the cells were stained with antibodies against CD3, CD4, CD8, PD-1, CD44, and CD62L on ice for 30 min. All samples were analyzed using a BD LSRII flow cytometer, and the data were analyzed using FlowJo software.
In detail, the antibodies used were anti-CD3-AF488 (cat. #100210; BioLegend), anti-CD4-AF700 (cat. #557956; BD Biosciences), anti-CD8-allophycocyanin (APC) (cat. #100712; BioLegend), anti-PD-1-PE (cat. #12-9969-42; eBioscience), anti-CD44-percpcy5.5 (cat. #103031; BD Biosciences), and anti-CD62L-BV421 (cat. #562910; BD Biosciences).

After the mice were sacrificed, the BALF was harvested and lysed by Ammonium-Chloride-Potassium Lysis buffer (Beyotime Institute of Biotechnology). To detect ROS generation, the sedimented cells were resuspended in PBS. Briefly, the cells were incubated with 50 µM of DCFH-DA for 30 min at 37˚C in darkness. DCF fluorescence intensities were measured by flow cytometry. At the end of the treatment, the cells were incubated with 10 µM dichloro-dihydrofluorescein diacetate (DCFH-DA; Beyotime Institute of Biotechnology) for 1 h at 37˚C in the dark. ROS generation in the cells was measured immediately using a fluorescence microscope (magnification, x200; Nikon Corporation). Myeloperoxidase (MPO) activity, malondialdehyde (MDA) content, superoxide dismutase (SOD) activity, and glutathione (GSH) content were also measured. The lung homogenate was dissolved in extraction buffer to detect the levels of MPO, MDA, SOD and GSH using commercially available assay kits (cat. nos. A044-1-1, A003-1-2, A001-3-2 and A005-1-2, respectively; Nanjing Jiancheng Bioengineering Institute), according to the manufacturer's instructions.