Cryopreserved PBMC Flow Cytometry

Cryopreserved PBMCs were thawed as previously described (37 (link)), and resuspended at 5 × 10⁶ cells/ml in PBS. Cells were incubated with 10% FCS at 4°C for 10 min prior to staining for 30 min with PerCP-Cy5.5 conjugated anti-CD3 (clone OKT3 from eBiosciences, San Diego, CA, USA). Cells were permeabilized with permeabilization buffer [made up of 0.1% NaAzide and 0.1% saponin in Dulbecco’s PBS (Lonza, Verviers, Belgium)], and incubated with FITC conjugated anti-CD3ζ antibody (clone 6B10.2 from BioLegend, San Diego, CA, USA). At least 50,000 live events were acquired on an LSR II flow cytometer (BD Biosciences, San Jose, CA, USA). Analysis was performed using FlowJo software (TreeStar, USA) and mean fluorescence intensity (MFI) was calculated for CD3 and CD3ζ. CD3ζ expression on T cells was determined according to expression levels on CD3 positive cells, and normalized by subtracting the MFI of CD3 in the CD3 negative population.