Immunophenotyping of the peripheral blood immune cells by flow cytometry

Deeply anesthetize the animal. Check for no reflexes and slower breathing

Cut the animal's thorax and collect peripheral blood from the left ventricle

Lyse the whole blood with the RBC (1X) solution for 10 minutes at 37°C.

Upon lysis, wash cells and resuspend in cold PBS for immunophenotyping

Stain cells surface with FITC-conjugated anti-CD4(1:100, Biolegend), anti-agranulocytes (1:100, Miltenyi biotec), PerCP5.5-conjugated anti-CD8 (1:100, Biolegend) or anti-CD45 (1:100, Biolegend), APC-conjugated anti-CD25 (1:80, Biolegend), PE-conjugated anti-CD45RA (1:100, Biolegend), APC-conjugated anti- CD161 (1:100, Biolegend), PE-Vio770 conjugated anti-CD3 (1:100, REAffinity Miltenyi biotec) and APC-Vio770-conjugated anti-CD11b/c (1:100, REAffinity Miltenyi biotec.) for 15 minutes at 4°C in the dark

Wash 5 minutes in cold PBS

Incubate with a fixing solution (1:3) for 15 minutes at room temperature in the dark

Wash 5 minutes in cold PBS

Permeabilize cells with Cytofix/Cytoperm (1:10, Biolegend) and then stain them intracellularly with PE-CF594-conjugated anti-FoxP3 (1:50, BD Biosciences) in Cytoperm at room temperature for 30 min

Acquire cells with an appropriate flow cytometer

Gate cells on CD45 to identify total leucocytes. Inside this gate, identify granulocytes with their specific marker, monocytes, and myeloid cells as CD11b/c+, T-lymphocytes as CD3+, B-lymphocytes as CD45RA+, and NK cells as CD161+ cells.

For each analysis, acquire at least 0.5x10^6 live by gating on aqua Live/Dead negative cells and then analyze using Flowjo software (Tree Star)