Hi,
I work with Leishmania parasites and would like to measure the free cytosolic Calcium concentration in them. For this I plan to use Fluo 4-AM dye from Thermo. As I see mostly people use some sort of plate reader to meassure the fluorescence intensity of the dye. As I understood one crucial step of assays that involve Flu 4-AM is that you need to wash the cells before reading the fluorescence intensity, otherwise the unbound free dye in the incubation media would change the reading.
Specifically for my case these parasites are motile and following incubation if I want to wash the cells, I might loose some cells from individual samples (which might differ from sample to sample), and as a result of which I might end up having different number of cells for different samples.
I was wondering if I could use flow cytometry with Fluo 4-AM dyes? Typically we measure mean fluorescence intensity from 10000 cells/events in a flow cytometer. Since my starting cell populations are typically much higher thatn this number, so even if I loose some cells, I would still have enough sample to run through a flow cytometer.
You comments would be much appreciated!
Sumit
It has been done. Here is an example:
http://onlinelibrary.wiley.com/doi/10.1002/cyto.a.20974/full
There is only one pre-caution for such measure: dye is not ratiometric. So, you must be care about differences in dye uptake and de-esterification in different cell lines/after treatments. .
I used the ratio of Fluo-4/Fura Red to quantified calcium release upon stimulation of cells with IgM by flow cytometry. That way you get a ratiometric quantification. Having a positive control, in our cases Thapsigargin, is a good way to know what good signal is supposed to be.
Dear Sumit,
An alternative is to immobilize parasites by attaching them to polylysine-precoated slides. This usually works fine for fluorescence microscopy.
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