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).