The pellet of BALF cells was resuspended in 300 µL of PBS with bovine serum albumin (BSA) and used for flow cytometry analysis. The cells were pipetted through cell strainer cap directly to the well of 96-well plate and were centrifuged (5 min, 1500 rpm, 4℃). Then supernatant was discarded and cells were resuspended in 54 µL of 5% normal rat plasma in PBS with BSA and blocked for 15 minutes on ice. Then, the cells were divided into two parts and to each 3 µL of antibody cocktail was added (according to Table 2 ). The cells were stained on ice for 30 minutes in dark, washed twice in PBS with BSA and resuspended in PBS with BSA and NaN3 for the flow cytometry analysis (Guava, Merck).
Antibodies Used for Flow Cytometry Analysis
| Antibody | Cat. No. |
|---|---|
| Anti-CD11b-AF488 (clone M1/70) | Biolegend, 101217 |
| Anti-CD45.2-PE (clone 104) | eBiosciences, 12–0454 |
| Anti-Ly6G-PerCP-Cy5.5 (clone 1A8) | Biolegend, 127616 |
| Anti-CD11c-APC (clone N418) | eBiosciences, 17–0114–82 |