Percp cy5.5 anti cd8a

Mouse splenocytes were blocked with anti-CD16/CD32 mAbs and stained with eFluor 450 viability dye (93: catalog 101320, BioLegend). Antibodies used for cell surface proteins were Alexa Fluor 700–anti-CD3 (17A2: catalog 100215), PerCP-Cy5.5–anti-CD8a (57-6.7: catalog 100734), APC-Cy7–anti-CD4 (GKq.5: catalog 100412), PE-Cy7–anti-CD19 (6D5: catalog 115520), PE–anti-CD62L (W18021D: catalog 161203), PE-Cy7–anti-CD44 (IM4: catalog 103024) (all BioLegend), and APC–anti-CD11c (3.9: catalog 17-0116-42, Thermo Fisher Scientific). The cells were then stained with fluorescence antibodies or H-2D(b) SARS-CoV-2 N_219-227 LALLLLDRL (Alexa Fluor 647–Labeled Tetramer) or H-2K(b) SARS-CoV-2 S_539-546 VNFNFNGL (Alexa Fluor 647–Labeled Tetramer) (NIH Tetramer Facility, Emory University). For intracellular staining, the cells were permeabilized in PBS containing 0.1% saponin and fixed for 10 minutes in 4% paraformaldehyde. The cells were then stained with PE–anti–IFN-γ (XMG1.2: catalog 505808), APC-cy7–anti–TNF-α (MP6-XT22: catalog 506343), PE–anti-perforin (S16009A: catalog 154305), and PE-cy7–anti-granzyme (QA16A02: catalog 372213) (BioLegend). The cells were analyzed on an LSR II flow cytometer (BD) and the data were analyzed with FlowJo software.

For surface staining, PBCs were incubated with fluorochrome-labeled antibodies specific for humans before fixation: AF-700-anti-CD45 (2D1), percp-anti-CD19 (HIB19), APC/CY7-anti-CD3 (UCHT1), BV510-anti-CD4 (OKT4) and percp/Cy5.5-anti-CD8a (HTT8a). Antibody-stained PBCs were fixed overnight with 4% PFA at 4 °C and taken out of BSL3 lab for downstream analysis. Cells were stained further with in-house-made SARS-CoV-1 NP pAb (1:500) at 4 °C for 30 min after permeabilization. Then cells were stained with FITC-anti-Rabbit IgG (H + L) at room temperature for 30 min. AF-700-anti-CD45 (2D1), APC/CY7-anti-CD3 (UCHT1), BV510-anti-CD4, and percp/Cy5.5-anti-CD8a antibodies were purchased from Biolegend and all were used at 1:100. FITC-anti-Rabbit IgG (H + L) was from Proteintech (SA00003-2).