Calcium Flux Protocol
- Wash cells 3 times in room temperature HBSS (Hank's Buffered Salt Solution) at pH 7.1
- Be sure there is no Calcium in the buffer, as the presence of Calcium can affect loading
- Cells should be happy and in ~mid log phase- don't use them if they've already exhausted their media
- Spin cells at 1050 RPMs in swing bucket centrifuge (~500 xg) for 3-5 minutes
- Cells stay happier if round-bottom tubes are used during the spins
- Count cells and dilute to 2 million per mL prior to loading
- Load cells with dye (indo 1-AM, molecular probes, 6µg/mL, 40 minutes at 37°C, 5% CO2)- varies significantly by cell type; determine experimentally
- Be sure to mix tube by inversion every 10 minutes to ensure even loading
- Wash loaded cells 3 times in fresh FACS buffer (1X DPBS pH7.1, 1%(w/v) BSA, 1 mM CaCl2)
- BSA varies significantly by manufacturer and sometimes precipitates. Biotechnology Grade BSA from Research Organics works well.
- Prepare CaCl2 fresh and dilute from a fresh 1 M stock
- Never add sodium azide to this buffer to extend its shelf life- sodium azide will cause some cells to flux even at a final concentration (in the flow tube) as low as 0.0002%
- This buffer is best if made up fresh and also sterile filtered.
- Resuspend cells in the same FACS buffer to a final concentration of 1 million cells/mL
- Filter cells before running and add 5µL concentrated PI
- Usually filter 600 µL of cells and then put filtered 500 µL into a new flow tube (so you have an accurate volume)
- Run the experiment
Protocol courtesy of Erik Puffer (2/10/03).