Fc blocking buffer

Surface markers on fresh airway cells were labeled prior to fixation for optimal labeling as described previously [12 (link)]. Metal-conjugated antibodies (Table S1) for labeling cells were purchased (Fluidigm, Markham, ON), or carrier-free antibodies were conjugated in house using MaxPar X8 labeling kits according to manufacturer’s instructions (Fluidigm). EPX antibody was the generous gift of Elizabeth Jacobsen, PhD from the Mayo Clinic, Scottsdale, AZ [22 (link)]. Briefly, cells were washed with CyFACS buffer (PBS with 0.1% BSA, 2 nM EDTA, 0.05% Na azide; Maxpar® Cell Staining Buffer; Fluidigm, South San Francisco, CA), resuspended in 5 μM cisplatin (Enzo Life Sciences) for 5 minutes, and quenched with 100% FBS. Samples were incubated with 11% Fc blocking buffer (Biolegend, Dedham, MA), surface labeled for 30 min on ice, fixed (BD FACS Lyse), and frozen at -80°C. Intracellular labeling was conducted on batches of samples (10–20/day). Fixed cells were permeabilized (BD FACS Perm II) for labeling with intracellular antibodies for 45 min on ice. Cells were suspended overnight in iridium interchelator (125 nM; Fluidigm) in 2% paraformaldehyde in PBS and washed 1X in PBS and 2X in H2O immediately before acquisition. Samples were run on Helios 2 CyTOF instrument at a flow rate of 30 μL/min and a minimum of 100,000 events was collected.