HI,

I study lipid metabolism in Leishmania parasites and have generated some lipid mutants. I would like to measure the cytosolic calcium in these mutants and compare them with that of the wild type. I see that often time people use either spectrofluorometer or fluorescence plate reader or flow cytometer for this purpose. We have flow cytometer and I would like to use that. I am a bit confused with the protocol though. I have purchased the Fluo-4 AM dye (cell permeable calcium indicator) and pluronic F-127 (use to help dispersion of  FL4-AM). My questions are:

1. Do I add both FLuo-4 AM and Pluronic F-127 at the same time and then incubate the cells?

2. I see for negative control people use Ca chelator (such as EGTA). Do I add EGTA along with the above two reagents at the same time or I should wait a while after adding EGTA and then add FLUO-4 AM and F-127?

3. Do I need any type of positive control? If yes, what would than be?

4. How the normalization is typically done?

I would greatly appreciate any comment or suggestion on this topic!

Thanks in advance!

Sumit