CALCIUM PROTOCOLS
>>>>>Indo-1-AM : purchased from Molecular Probes as 50mg in each of 20 vials (1-800-438-2209 cat # I-1223, approx price $128) mwt 1010. Store in freezer.
When needed, add 50ml of DMSO to the 50mg in one vial.
Dissolve well ---> 1mM stock solution.
For cell incubation, dilute 5ml of the 1mM stock into 1 ml of serum free medium ---> 5mM final concentration for incubation. Incubate cells (at about 107/ml) in this 5mM indo for 30 min at 37 degrees.
Wash cells well in calcium-containing medium; then keep cells at room temperature before use. Cells will probably be OK for several hours if kept at room temperature(indo leaks out rapidly at 37 degrees).
The 1mM stock solution of indo in DMSO can be kept in the freezer and used again for several months.
>>>>>Pluronic detergent: is given away free of charge by Molecular Probes. Can be useful for loading difficult cells, eg CHO cells or others which have strong efflux pumps. Make up a 20% stock solution of pluronic in DMSO. If cells don't load well, add 1ml of this detergent stock to 1 ml of cell suspension being loaded with indo (as above) to give a final 0.02% detergent solution.
>>>>> Ionomycin, free acid purchased as 1 mg from Calbiochem. mwt 709.
Add 357 ml of DMSO to 1 mg of ionomycin ----> 4mM stock solution. Store in freezer.
For ACAS:
When needed, add 5ml of ionomycin stock to 5 ml of reaction medium ----> 4mM solution.
Dilute 1:2 for by addition to cells in chamber on ACAS ----> 2mM final concentration.
For flow:
When needed, add 5ml of ionomycin stock to 45 ml of reaction medium ---> 0.4 mM solution.
Dilute 1:200 by addition to flow sample ----> 2 mM final concentration.