Code for analysing intracellular pH on a single cell level

Cells were plated in triplicate at 50,000-100,000 cells per well in black wall, flat coverslip bottom µ-plate 96-well plates with a growth area of 0.56 cm2 per well (Ibidi) and were left to attach overnight. They were then incubated in media supplemented with cSNARF1-AM (5 µg/mL, Invitrogen) and the nuclear stain Hoechst-33342 (10 µg/mL, Molecular Probes), for 15 min, and then replaced with medium of varying sodium bicarbonate concentration (twice). Images of fluorescence excited at 377 nm and collected at 447 nm (Hoechst-33342), and of fluorescence excited at 531 nm and collected at 590 nm and 640 nm (cSNARF1), were acquired using Cytation 5 imaging plate reader (Biotek) and its bespoke software. Images were either acquired using a 4x objective, or a 10x objective, as indicated in figure legends. Measurements were performed in an atmosphere of 37 °C and 5% CO2, established in the plate reader. Further analysis of the population distribution of pH data was performed using a MATLAB script. Images saved as stack.tif act as input for the script (supplementary_code_1). cSNARF1 fluorescence ratios were converted into pHi using a calibration curve obtained through the nigericin method. pHi distributions from replicate wells were pooled, and low intensity measurements were removed using a second MATLAB script (Supplementary_Code_2).