Immune Cell Profiling by Flow Cytometry

Splenocytes were treated with AECs for 48 h, collected in a 1.5‐ml centrifuge tube and then stained with primary antibodies, including anti‐rat CD3‐FITC, CD4‐PE, CD8‐APC, CD45‐RA+, CD161‐PE, CD80‐PE, CD86‐FITC, and CD103‐Alexa Flox@647 (eBiosciences, CA, USA). As reported, we defined the CD3⁺CD4⁺ population as helper T (Th) cells, CD3⁺CD8⁺ population as cytotoxic T (Tc) cells, CD3^‐CD45RA⁺ population as B lymphocytes, CD3^‐CD161⁺ population as natural killer (NK) cells, CD3⁺CD161⁺ population as natural killer T (NKT) cells, and CD80‐PE, CD86‐FITC, and CD103‐Alexa Flox@647 as dendritic (DC) cells (Ayako et al., 2018 (link); Chen et al., 2018 (link); Xu, Wusiman, et al., 2019 (link)). After incubation at 4°C for 30 min, the cells were washed twice and resuspended in PBS before they were transferred to fluorescence‐activated cell sorting (FACS) tubes and analyzed by flow cytometry (BD Biosciences, San Jose, CA, USA). The cells were gated using forward and side scatter for dead cell exclusion. In each sample, 10,000 events were measured, and data were analyzed using Flow Jo 7.6 software.