Percp cy5.5 conjugated ly 6c

Cells were collected from uninured and injured tissue digest, centrifuge at 400×g for 5 min, and were resuspended in PBS with 5% bovine serum albumin. Cells were then incubated with CD16/CD32 FcBlock (BD Biosciences, 553141) for 5 min at 4 °C. Cells were labeled using a 1:100 concentration of antibodies against APC-conjugated F4/80 (Thermo Fisher Scientific, MF48005), FITC-conjugated CD11b (Thermo Fisher Scientific, RM2801), PE-conjugated Ly6G (BD Biosciences, 551461), and PerCP-Cy5.5-conjugated Ly6C (eBiosciences, 45-5932) for 20 min at room temperature in dark and then fixed with fixative medium A (Thermo Fisher Scientific, GAS004) for 15 min at room temperature. Unstained and single-stained cells controls were used as compensation controls. Cells were then washed and resuspended in 500 μL of DPBS containing 0.5% fetal bovine serum and analyzed using FACS Calibur flow cytometer (BD Biosciences). The number of gastric F480⁺/CD11b⁺/Ly6C^hi/Ly6G^neg macrophages were quantified using FlowJo software according to our previously published protocol^{14 (link)}.