Cbd500

Stock solutions of barcoding dyes CBD 450 (BD Biosciences) and CBD 500 (BD Biosciences) were prepared as per the manufacturer's instructions in DMSO in polypropylene 96-well plates and stored at -80°C. Different combinations of the dyes were used to produce 16 barcoded cell populations resolved using final concentrations of CBD 450 (0.000, 0.015, 0.050, 0.150 mg/ml) combined with CBD 500 (0.000, 0.038, 0.125, 0.375 mg/ml; Figure S1). Fixed cells were washed with PBS and permeabilized in 100 µl ice cold methanol (Fisher Chemical) for 20 min at 2°C using a Biomek NX liquid handler. The barcoding dyes were diluted in ice cold PBS and 100 µl/well was added to the suspension of cells in methanol. The final concentration of DMSO from the barcoding dyes at this stage was 3.5%. The barcoding reaction was incubated in the dark for 30 min at 2°C and the cells were washed five times in ice cold FACS buffer (PBS with 0.5% bovine serum albumin (Sigma-Aldrich)). The barcoded cells from individual donors were pooled and pelleted.