2.3 Flow cytometry detection of SARS-CoV2 specific Tfh cells
Cryopreserved human PBMC were thawed and rested for 5h in 37℃. Then
1×106 cells per
well in duplicate were stimulated with 2µg/ml of SARS-CoV2 S peptides of
both wild type strain and delta strain (Milteni Biotech) for 20h. After
stimulation, cells were washed and stained with Zombie Dye Aqua
(Biolegend) followed by blocking by Trustain Fcx (Biolegend). Then they
were stained with a cocktail of monoclonal antibodies CD3 BUV 737(BD
Biosciences), CD4 PE (Biolegend), CCR7 APC (Biolegend), CD45RA BV 786
(Biolegend), OX-40 BV421(Biolegend), CCR6 BV711 (Biolegend), CXCR3 BV605
(Biolegend), CD25 BV650 (BD Biosciences), CXCR5 BUV 395 (BD Biosciences)
and Zombie Aqua dye (Biolegend) for live and dead cell discrimination.
Next, the cells were washed, fixed with 1% formaldehyde, and acquired
on a BD LSR Fortessa using BD FACS Diva. For intracellular cytokine
assay 1×106 cells per well in duplicate were
stimulated with 2µg/ml of SARS-CoV2 S peptides of both wild type strain
and delta strain (Milteni Biotech) for 20h with Brefeldin A being added
in the last 4hrs. Cells were then stained cocktail of monoclonal
antibodies CD3 BUV 737(BD Biosciences), CD4 PE (Biolegend), CCR7 APC
(Biolegend), CD45RA BV 786 (Biolegend), CXCR5 BUV 395 (BD Biosciences),
IFN-γ BV650(Biolegend), IL-17 BV711 (Biolegend), IL-21 BV 421 (BD
Biosciences), IL4 BV605 (Biolegend) and Zombie Aqua dye (Biolegend) for
live dead and dead cell discrimination. For positive controls cells from
some volunteers were treated with α-CD3 and α-CD28 for AIM assay and for
ICS they were stimulated with PMA/Ionomycin. Unstimulated cells were
used as negative controls.