Flow cytometry - Measurement of SARS-CoV-2 reactive T cells
As previously described, PBMCs were plated in 96-U-Well plates in RPMI
1640 media (Life Technologies) (22,23). Each well was stimulated with
wildtype (WT) S-protein (Miltenyi Biotec) or left untreated as a control
for 16 h. The proteins were dissolved per manufacturer’s directions. As
a positive control, cells were stimulated with staphylococcal
enterotoxin B (1 μg/mL, Sigma-Aldrich). After 2 h, brefeldin A (1 μg/mL,
Sigma-Aldrich) was added. Detailed listing of the antibody panels for
general phenotyping and T cell activation ex vivo is in Table S2.
The PBMCs stimulated overnight were stained with optimal concentrations
of antibodies for 10 min at room temperature in the dark. Stained cells
were washed twice with PBS/BSA before preparation for intracellular
staining using the Intracellular Fixation & Permeabilization Buffer Set
(Thermo Fisher Scientific) as per the manufacturer’s instructions. Fixed
and permeabilized cells were stained for 30 min at room temperature in
the dark with an optimal dilution of antibodies against the
intracellular antigen. All samples were immediately acquired on a
CytoFLEX flow cytometer (Beckman Coulter). Quality control was performed
daily using the recommended CytoFLEX daily QC fluorospheres (Beckman
Coulter). No modification to the compensation matrices was required
throughout the study. Antigen-reactive responses were considered
positive after the non-reactive background was subtracted, and more than
0.01% were detectable. Negative values were set to zero.