Cryopreserved Cell Staining for Cytokine Analysis

Cell staining was performed on cryopreserved fixed cells that were thawed and washed with 1% FACS washing buffer (1% FBS in PBS). A viability dye was not included during the staining process, as it has been reported that the omission of a viability dye during the staining process does not impact the detection and quantification of cytokines when using the whole blood-based T cell assay.⁸⁸ (link) Cells were stained with the following surface antibody markers: CD4 ECD (SFCI12T4D11, Beckman Coulter), CD8 BV510 (RPA-8, Biolegend) and incubated at room temperature for 20 min. Cells were permeabilized and stained with intracellular antibody markers CD3 BV650 (OKT3), IFN-γ AlexaFluor 700 (B27), TNF-α BV786 (Mab11) and IL-2 APC (MQ1-17H12) (all from Biolegend). Finally, cells were washed and fixed with Cellfix (BD Biosciences). Samples were acquired on a multiparameter BD Fortessa flow cytometer using Diva software version 9 and analyzed using FlowJo v10. Results are expressed as the frequency of CD4⁺ or CD8⁺ T cells expressing IFN-γ, TNF-α or IL-2. Cytokine responses presented are background subtracted values (from the frequency of cytokine produced by unstimulated cells).