The following protocol is for calcium flux using calcium green with fura red and single laser (488 nm) excitation.

Reagents

  • Calcium Green-1, AM (Molecular Probes cat# C-3012 [special packaging, 10 x 50µg], molecular weight = 1290.98
  • Fura Red, AM (Molecular Probes cat# F-3021 [special packaging, 10 x 50µg], molecular weight = 1089.00

Buffer #1

  • 25mM Hepes at pH 7.2 (2.5ml of a 1M stock)
  • 125mM NaCl (0.73g)
  • 5mM KCl (0.037g)
  • 1mM Na2HPO4 (.014 g)
  • 0.1% Glucose (Dextrose) (0.1g)
  • QS to 100ml with dH2O and store at 4°C

Buffer #2

Just prior to use:

  • 1ml MgCl2 (50mM stock)
  • 1ml CaCl2 (0.1M stock)
  • 0.1g BSA
  • QS to 100ml with buffer #1 pH to 7.4 and keep on ice

Staining

  1. Dissolve 50µg of each dye in 100µl DMSO
  2. Suspend cells in 1.5mls of buffer #2 at 5 - 7 x 106/ml in a 15ml conical tube
  3. Add 20µl of each dye per ml of cell suspension
  4. Incubate 30 minutes at 37°C in the incubator
  5. Wash one time in 14ml buffer #2
  6. Resuspend at a concentration of 1 - 2 x 106/ml in buffer #2
  7. Immediately place on ice
  8. Warm to 37°C before running on flow cytometer
  9. Use the green channel (530/30 nm optical filter) for calcium green and far red channel (660/20 or 670/30 nm optical filter) for fura red in linear mode. Use FlowJo or other software to calculate the ratio of calcium green (bound) over fura red (unbound).