Tnf α bv650

PBMCs were cultured in RPMI-1640 media (GIBCO, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS), with or without LPS (100 ng/mL, STEMCELL Technologies, Vancouver, Canada) and Golgiplug (BD Biosciences, San Diego, CA, USA) for 3 h. The cells were surface-stained with CD45-BV786, CD14-Alexa Fluor 700, CD16-BV711, HLA-DR-APC-H7, TIM-3-BB515 (BD Biosciences, San Diego, CA, USA), and TIGIT-PE-Cy7 (eBioscience, San Diego, CA, USA), and intracellularly stained with antibodies against IL-10-APC, IL-1β-Pacific blue, TNF-α-BV650 (BD Biosciences), IL-6-PE (eBioscience), GM-CSF-PE-CF594 (BioLegend), and the corresponding isotype controls. Data acquisition was performed on an LSR Fortessa flow cytometer (BD Biosciences, San Diego, CA, USA), and data were analyzed with FlowJo software (Tree Star, Ashland, OR, USA).

For the intracellular cytokine staining, mouse spleen cells were stimulated with 2 μg/mL S1, S2 peptide pools spanning SARS-CoV-2 spike S1 and S2 respectively (15mers, overlapping by 11aa, GenScript, Nanjing, China) or equimolar amount of DMSO (negative control) in the presence of anti-mouse CD28 antibody (BD Bioscience, CA, USA) for 1 h. GolgiStop protein transport inhibitor (BD Bioscience) was added into the culture and further incubated for 5 h. After stimulation, cells were washed and stained with Fixable Viability Stain 510 (BD Bioscience). Cells were then blocked with anti-mouse CD16/32 and labeled with cell surface antibody cocktail including anti-mouse CD3-FITC, anti-mouse CD4-APC and anti-mouse CD8-Percp-cy5.5 (BD Bioscience) for 30 min at 4°C. Following surface staining, cells were incubated with fixing/permeabilizing solution (BD Bioscience) for 20 min at 4°C and stained with intracellular antibodies including anti-mouse IFN-γ-Pe-Cy7, IL-2-BV605, TNF-α-BV650, IL-4-BV711, and IL-5-PE (BD Bioscience) for 30 min at 4°C. Cells were washed and resuspended in PBS buffer and analyzed on a CYTEK Aurora/NL.