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.