American Family Children's Hospital

Calcium Flux Protocol

  1. 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
  2. Count cells and dilute to 2 million per mL prior to loading
  3. 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
  4. 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.
  5. Resuspend cells in the same FACS buffer to a final concentration of 1 million cells/mL
  6. 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)
  7. Run the experiment

Protocol courtesy of Erik Puffer (2/10/03).