Apc labeled anti mouse cd4

Freshly isolated splenocytes or peripheral blood mononuclear cells were plated into round-bottom 96-well plates (2×10⁶ cells per well) and incubated with either R10 (RPMI1640 with 10% FBS) or R10 containing synthesized peptides encompassing the full length of S protein (0.66μg/ml for each peptide) (Synthesized by Gill Biochemistry Co., Ltd., Shanghai, China). Two hours later, brefeldin A and monensin were added to each well at final concentrations of 1μg/ml and 1μM, respectively. Another 12 hours later, the cells were washed and stained sequentially with Live/Dead dye (Fixable Viability Stain 510, cat# 564406, BD Pharmingen), surface markers (PE/Cyanine7-labeled anti-mouse CD3, cat# 100220, BioLegend; APC-labeled anti-mouse CD4, cat# 100412, BioLegend; PE-labeled anti-mouse CD8, cat# 100708, BioLegend) and intracellular markers (BV421-labeled anti-mouse IFN-γ, cat# 505830, BioLegend; FITC-labeled anti-mouse IL-2, cat# 503806, BioLegend; BV711-labeled anti-mouse TNF-α, cat# 506349, BioLegend). Stained samples were analyzed using a BD Fortessa flow cytometer and data were analyzed with the FlowJo software version 10 (TreeStar Inc., Ashland, OR, USA). The gating strategy was shown in Supplementary Figure 1.