Functional analysis of stimulated PBMC by mass cytometry
All antibodies used in the study were labelled in house with metal tags. Purified antibodies for mass cytometry were obtained in carrier/protein-free buffer and coupled to lanthanide metals using the MaxPar DN3 antibody conjugation kit (Fluidigm Inc.) according to the manufacturer’s recommendations. Upon conjugation, the yield of recovered antibody was determined by measurement of absorbance at 280 nm on a nanodrop before dilution of the antibodies in Candor PBS Antibody Stabilization solution (Candor Bioscience, Germany) for long-term storage at 4°C. Antibodies used are listed in Supplementary Table 1 . In order to capture the phenotype of resting cells, most of the surface markers were stained prior to the stimulation. Frozen PBMCs were thawed using RPMI 1640 Medium (Gibco), supplemented with 10% FBS (BioWhittaker) and washed with homemade 1X MACS buffer. Cells were resuspended in pre-stimulation antibody cocktail and incubated for 30 minutes on ice. Cells were washed with CyFACS buffer (PBS with 4% FBS, 0.05% sodium azide) and transferred to a 6-well plate for stimulation. The cells were stimulated using the PMA/Ionomycin cocktail described earlier and incubated for 4 hours in a 37°C incubator. The cells were then transferred into a 96-well U bottom plate and washed with PBS, followed by post-stimulation surface antibody cocktail and incubation of 30 minutes at 37°C. Subsequently, cells were washed with CyFACS buffer and resuspended with the post-stimulation antibody cocktail and incubated for 30 minutes on ice. Cells were washed twice with CyFACS buffer and then treated with Cisplatin for 5 minutes on ice. Following double washes with CyFACS buffer and fixation overnight using 2% PFA, cells were permeabilized and stained with intracellular antibody cocktail for 30 minutes on ice. Washes were then carried out with permeabilization buffer followed by barcoding. Lastly, cells were stained with DNA intercalator (Cell-ID Intercalator-Ir, Fluidigm) in PBS for 20 mins at room temperature. After washing twice with CyFACS buffer, cells were washed with MiliQ water and filtered through a cell strainer snap cap tube in preparation of CyTOF acquisition. Cells were then diluted to 0.5 × 106 cells/mL in MiliQ water containing 2% EQ™ Four Element Calibration Beads (Fluidigm). Cells were acquired using Helios mass cytometer at a rate of 280-350 cells/s on CyTOF software version 7.0.5189 and analysed using FlowJo v10 (BD).