Flow Cytometry Assays
We followed previous research method to perform flow cytometry assay27. For isolation of splenocytes, spleen was mechanically dispersed through a 100μm cell-strainer and washed with PBS. The cellular suspension was then centrifuged at 1500r for 10 min at 4°C, then incubated with Red Blood Cell Lysis Buffer (Solarbio, # R1010) for 5 min in the dark. Afterward, samples were centrifuged at 1500 RCF for 10 min at 4°C, then washed with PBS and re-suspended in 1 mL PBS. The obtained cellular suspension was stained with fluorescent antibodies.
Before immunofluorescent staining, the cellular suspension of splenocytes was incubated with Fc (Biolegend, #101319) for 10 min in the dark at 4°C, in order to block non-specific binding sites for antibodies. The following antibodies were used:CD11b (FITC, Biolegend, # 101205), F4/80(Percpcy5.5, Biolegend, #123127), CD206(PE, Biolegend,#141705), CD86(APC, Biolegend,#105011), CD4(FITC, Biolegend, #100405), CD25(APC, Biolegend, #102011), Foxp3(PE, Biolegend, #126403), CD3(FITC, Biolegend, #100203), NKp46(PE, Biolegend, #137603).
Samples were analyzed using CytExpert software. The spleen cell populations were defined as follows: Treg cell (CD4+CD25+FoxP3+), M1 macrophage (CD86+F4/80+CD11b+), M2 macrophage (CD206+F4/80+CD11b+), NK cell (CD3-NKp46+). Percentages were reported to the total number of splenocytes or MLN cells of each mouse to calculate the number of cells per population.