Ic fixation permeabilization kit

PBMCs were resuspended at 10x10⁶ cells/mL RPMI (Gibco by Life Technologies) supplemented with penicillin/streptomycin (Gibco by Life Technologies), 10% heat inactivated FCS, and incubated at 37°C, 5% CO₂. After a rest of 2hrs, cells were stimulated with 0.5 μg/mL staphylococcal enterotoxin B (SEB) or 0.5 μg/mL of overlapping peptide pools for Wuhan-1 or Omicron BA.1 variants SARS-CoV-2 Spike (JPT) for 6 hrs at 37°C, 5% CO₂. An unstimulated condition with 0.4μL of DMSO served as a negative control. Brefeldin A (BD Biosciences), Monensin-1 (BD Biosciences), and a fluorescently labeled CD107a antibody were added for the remaining 5hrs.
Cells were stained for viability dye (Aquavivid, Thermofisher, 20min, 4°C), surface markers (30min, 4°C), and intracellularly for cytokines (30min, room temperature) using the IC Fixation/Permeabilization kit (eBioscience) (see Table S4 for antibodies) and filtrated before acquisition on the flow cytometer (FACSymphony A5 Cell Analyzer, BD Biosciences) and analyzed using FlowJo (BD, v10.6.2).

The ICS assay adapted to study SARS-CoV-2-specific T cells was previously described (Tauzin et al., 2021b (link)). PBMCs were thawed and rested for 2 h in RPMI 1640 medium supplemented with 10% FBS, Penicillin-Streptomycin (Thermo Fisher scientific, Waltham, MA) and HEPES (Thermo Fisher scientific, Waltham, MA). 1.7×10⁶ PBMCs were stimulated with a S glycoprotein peptide pool (0.5 μg/mL per peptide from JPT, Berlin, Germany) corresponding to the pool of 315 overlapping peptides (15-mers) spanning the complete amino acid sequence of the S glycoprotein.
Cell stimulation was carried out for 6h in the presence of mouse anti-human CD107a, Brefeldin A and monensin (BD Biosciences, San Jose, CA) at 37 °C and 5% CO₂. DMSO-treated cells served as a negative control, and SEB as positive control. Cells were stained for Aquavivid viability marker (Thermo Fisher scientific, Waltham, MA) for 20 min at 4 °C and surface markers (30 min, 4 °C), followed by intracellular detection of cytokines using the IC Fixation/Permeabilization kit (Thermo Fisher scientific, Waltham, MA) according to the manufacturer’s protocol before acquisition on a Symphony flow cytometer (BD Biosciences) and analysis using FlowJo v10.8.0 software. Abs used are listed in the Table S3.

For the detection of some cytokines (IFNγ, IL-2, TNFα, IL-21, CXCL13) within AIM⁺ Ag-specific CD4⁺ T cells, cultured PBMCs were incubated with a CD40-blocking antibody, stimulated with peptide pools for 9 h as described above and further incubated for 12 h at 37 °C in the presence of Brefeldin A (BD Biosciences, Cat# 555029). Cells were surface stained, fixed and permeabilized using the IC Fixation/Permeabilization kit (Thermo Fisher, Cat# 88-8824-00) and incubated with antibodies against cytokines for 30 min at 4 °C (see Table S4 for antibodies).