L34968

The cryopreserved PBMCs from healthy controls and COVID+ subjects were rapidly thawed, washed twice in pre-warmed complete RPMI-1640 medium (Cytiva; SH30027FS, supplemented with 10% heat-inactivated human serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine and 20 mM HEPES), and suspended in 500 μl pre-warmed complete medium. The viability was assessed using trypan blue stain (BioBasic Inc. NY, US) and flow cytometry-based Live/dead staining (L34968, Thermo Fisher Scientific, CA, US). 1/5^th of the total cells were used for ex vivo staining or polyclonal stimulation with PMA and ionomycin (PMA-50 ng/ml and ionomycin-500 ng/ml) for 4 h followed by the addition of Brefeldin-A (10 μg/ml) in the last 2 h. The remaining cells were stimulated with PepTivator^® SARS-CoV-2 Prot_S Complete (130-127-953, Miltenyi Biotec), which is a pool of lyophilized peptides covering the complete protein coding sequence of the SPIKE glycoprotein of SARS-CoV-2 and capable of stimulating T cells. PBMCs were stimulated with these peptides (1 μg/ml) in the presence of IL-2 (50 U/ml) for 12 h followed by the addition of Brefeldin-A (10 μg/ml) in the last 2 h. Following stimulation, the cells were harvested, fixed and used for intracellular flow cytometry.

Anti CD133 (ab222782; 1:1000) and anti Nestin (ab134017; 1:500) antibodies were purchased from Abcam (Cambridge, MA). Anti Sox2 clone D6D9 (3579; 1:1000) and anti Oct-4A clone C30A3 (2840; 1:500) antibodies were purchased from Cell Signaling (Danvers, MA). Anti L1CAM (AF277; 1:1000) and anti-NFASC (AF2325; 1:500) were purchased from R&D Systems (Minneapolis, MN). Anti NCAM2 (sc-136328; 1:500) was purchased from Santa Cruz Biotechnology (Dallas, TX). Anti β-actin clone AC-40 (GTX11003; 1:10000) was purchased from Genetex (Irvine, CA). Anti CD11b/c clone OX-42 (201807; 1:100) was purchased from BioLegend (San Diego, CA). Anti CD133 clone TMP4 (46-1338-42: 1:200) antibody, Live/Dead fixable red stain (1:200; L34972) and Live/Dead fixable yellow stain (1:200; L34968) were purchased from Thermo Fisher Scientific. Secondary anti-rabbit IgG HRP-linked (7074; 1:10000) and anti-mouse IgG HRP-linked (7076; 1:10000) antibodies were purchased from Cell Signaling. Recombinant bFGF (233-FB) and EGF (236-EG) were purchased from R&D Systems (Minneapolis, MN).