The University of Iowa Carver College of Medicine

Flow Cytometry Facility

Calcium Flux Using
    Calcium Green and Fura Red

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 weigh t = 1290.98
  • Fura Red, AM (Molecular Probes cat# F-3021 [special packaging, 10 x 50µg], molecular weigh t = 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 14mls 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 (FL1 on FACScan) for calcium green and far red channel (FL3 on FACScan) for fura red in linear mode. Use FlowJo or other software to calculate the ratio of calcium green (bound) over fura red (unbound).