Fluorescence activated cell sorting lysing solution

To analyze cytokine-producing T cell populations, the lung lymphocytes were resuspended in IMDM supplemented with 10% heat-inactivated fetal bovine serum (10% IMDM) and stimulated with 10 μg of synthetic cryptic-epitope peptide (LSMPFHNIHPLTIGECPKYVKSVR) in 10% IMDM containing 10 ng of recombinant human IL-2 (BioLegend, San Diego, CA, USA) and Brefeldin A (1:1000; Thermo Fisher Scientific, Waltham, MA, USA) at 37°C for 5 h in dark. After stimulation, the cells were incubated with rat anti-mouse CD16/CD32 (BD Biosciences) blocking antibody for 10 min at room temperature, and then, the lung cells were stained with anti-CD4 (RM4-5; BioLegend) and anti-CD8 (53-6.7; BioLegend) antibodies for 30 min at 4°C in the dark. For intracellular cytokine staining, the stained cells were fixed with fluorescence-activated cell sorting lysing solution (BD Biosciences) for 20 min at room temperature, and permeabilized with fluorescence-activated cell sorting buffer (0.5% fetal bovine serum, 0.09% NaN₃ in PBS) containing 0.5% saponin (MilliporeSigma) for 15 min at room temperature. Then, those cells were stained with anti-IFN-γ (XMG 1.2; BioLegend) for 30 min at room temperature in the dark. The stained cells were analyzed by LSR Fortessa (BD Biosciences), and all flow cytometry data were analyzed by FlowJo software (Tree Star, Ashland, OR, USA).