How to measure intracellular pH using flow cytometry?

RatioWorks™ BCFL, AM (21190) is a cell-permeable fluorogenic probe that can be used in flow cytometric assays to measure intracellular pH. Below is a brief protocol:

1. Prepare cells for flow cytometry – for optimal performance flow cytometry requires between 2 x 10⁵ to 1 x 10⁶ cells
2. Centrifuge to remove growth medium
3. Add 1X 100 nM RatioWorks™ BCFL, AM working solution
4. Incubate at 37 °C for 30 to 60 minutes
5. Centrifuge to remove the dye loading solution
6. Re-suspend cells in 1 mL Hank's Buffer with Hepes (HHBS).
7. Analyze your sample with a flow cytometer using FITC channel