Anti mouse cd4 clone gk1

Lymph nodes were homogenized in RPMI-1640 culture medium with 10% fetal bovine serum, 100 U/ml penicillin/streptomycin, 2 mM L-glutamine, 1x MEM non-essential amino acids (Gibco, USA), 1 mM sodium pyruvate (Corning, USA), 10 mM HEPES (Fisher Scientific, USA), and 40 µM 2-mercaptoethanol (Sigma Aldrich) in gentleMACS C tubes (Miltenyi, USA). 0.5x10⁶ cells were incubated for 4 h with 20 ng/ml phorbol 12-myristate 13-acetate (PMA), 1 µg/ml ionomycin (Sigma Aldrich), and 3 µg/ml brefeldin A (Invitrogen, USA).
Subsequently, cells were washed with stain buffer containing bovine serum albumin (BD Pharmingen, USA) and stained with anti-mouse CD4 (clone GK1.5) and anti-mouse CD8a (clone 53-6.7, BioLegend, USA). Cells were then fixed, washed, and stained with anti-mouse IFN-γ antibody (clone XMG1.2, BioLegend), anti-mouse IL-4 antibody (clone BVD4-1D11, Miltenyi), or anti-mouse IL-17A antibody (clone TC11-18H10.1, BioLegend). Cells were analyzed on a Novocyte 3005 flow cytometer (Acea Biosciences, USA). The following gating strategy was used in FlowJo software v.10.6.0 (FlowJo, USA): size (FSC-H vs. SSC-H) → single cells (FSC-H vs. FSC-A) → CD8a+ vs CD4+ → IFN-γ+ (Th1), IL-4+ (Th2), or IL-17A+ (Th17) of the CD4+CD8- population.