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Monitoring Receptor-Mediated Changes in [Ca2+]i Using Single-Wavelength and Dual-Wavelength Indicators on the FlexStation Workstation

t # 15630-056), FBS: (Fetal Bovine Serum, heat inactivated, Invitrogen Life Technologies, cat # 10099-141), Geneticin (G418, Invitrogen Life Technologies, cat # 10131-019).


Fluo-3 and Fura-2 reagent preparation
Step 1 Wash buffer: this buffer contains a final concentration of 1X HBSS and 20 mM HEPES diluted in sterile d.H2O. Important: To prevent dye extrusion by the anion exchange protein, the wash buffer is supplemented with 2.5 mM probenecid.

Step 2 Prepare a fresh stock of 250 mM probenecid. Dissolve 710 mg probenecid in 5 mL of 1 M NaOH then add 5 mL of wash buffer and agitate vigorously.

Step 3 Fluo-3 and Fura-2 stock solutions. Add 25 L DMSO to 50 mg Fluo-3 AM or Fura-2 AM (final concentration ~2 mM).

Step 4 Dye loading buffer. Mix 25 L Fluo-3 or Fura-2 stock solutions and 25 L 20% pluronic acid solution, add this to 12 mL wash buffer containing 2.5 mM probenecid then add 120 L FBS. This prepares enough reagent for approximately one 96-well plate. Pluronic acid is used to enhance dye solubility and uptake.


Calcium assay kit reagent preparation
Step 1 Prepare 1X Reagent Buffer using component B and Loading Buffer by diluting component A with 1X Reagent Buffer as described in the instructions provided with the kit

Step 2 Prepare a fresh stock of 250 mM probenecid as above.

Step 3 Add 1:50 dilution of 250 mM probenecid to the Loading Buffer (final concentration 5 mM) and 1:100 dilution of 250 mM probenecid to 1X Reagent Buffer (final concentration 2.5 mM).


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