Intracellular Staining and Mass Cytometry of Cryopreserved Splenocytes

For intracellular staining, short-term reactivation of cryopreserved splenocytes and subsequent mass cytometry analysis were performed as described previously^{94 (link)}. In short, cells were kept at −80 °C for less than 2 months before thawing in a 37 °C water bath. Cells were then immediately resuspended in cell culture medium supplemented with 1:10,000 benzonase (Sigma-Aldrich), and centrifuged at 300 g for 7 min at 24 °C. Samples were then left overnight at 37 °C before restimulation with 50 ng/mL phorbol 12-myristate 13-acetate (Sigma-Aldrich) and 500 ng/mL ionomycin (Sigma-Aldrich) in the presence of 1× brefeldin A (BD Biosciences) and 1× monensin (Biolegend) for 4 h at 37 °C. For splenocytes, one anchor sample to correct batch effect among different acquisitions and one non-stimulated control sample were also included. For both PBMCs and reactivated cryopreserved splenocytes, surface staining was performed for 30 min at 4 °C. To identify dead cells, 2.5 μM cisplatin (Sigma-Aldrich) was added for 5 min on ice. To minimize technical variability, an equal number of cells from each sample were barcoded using Cell-ID 20-Plex (Fluidigm). Cells from all samples were then combined in one single tube. The combined sample was then processed for Live/Dead and surface staining. For intracellular cytokine staining of reactivated cryopreserved splenocytes, after surface staining, cells were fixed and permeabilized with FOXP3 staining kit (Invitrogen) and stained for intracellular markers and cytokines. The antibodies used are reported in Supplementary Table 2. Because CD4 molecules are internalized during phorbol 12-myristate 13-acetate/ionomycin stimulation^{95 (link)}, the anti-CD4 antibody was used in both the surface staining and the intracellular staining steps. After washing, the combined sample was incubated with 4% PFA overnight at 4 °C. Prior to acquisition in a Helios™ II CyTOF® system, samples were washed with cell staining buffer and mass cytometry grade water. Multidimensional datasets were analyzed using FlowJo and R (R Core Team, 2017).