PMA induced apoptosis in HEK cells - (Jul/22/2007 )

I will be inducing apoptosis in HEK293 cells (transfected with a fluorescent protein) with PMA (0.01uM-10uM) then passing them through a flow cytometer to analyse levels of apoptosis. I plan to trypsinise my cells, spin, resuspend in FACS buffer and to this add the relevant concentration of PMA, incubate for 20-30 minutes and then analyse forward and side scatter by flow cytometry. Does this sound like a suitable protocol? I know that PMA is very potent so are my incubation times too long or my concentrations too high for this purpose? As I don't want to kill my cells before analysis, I want to see a gradual effect by increased PMA concentrations.

Any tips will be really appreciated!

Maybe the exporsure period is too short? Why not treat the cells for 2 to 4 hrs then trypsinize and do the analysis?

I will be inducing apoptosis in HEK293 cells (transfected with a fluorescent protein) with PMA (0.01uM-10uM) then passing them through a flow cytometer to analyse levels of apoptosis. I plan to trypsinise my cells, spin, resuspend in FACS buffer and to this add the relevant concentration of PMA, incubate for 20-30 minutes and then analyse forward and side scatter by flow cytometry. Does this sound like a suitable protocol? I know that PMA is very potent so are my incubation times too long or my concentrations too high for this purpose? As I don't want to kill my cells before analysis, I want to see a gradual effect by increased PMA concentrations.

Any tips will be really appreciated!

QUOTE (Debz @ Jul 22 2007, 09:57 PM)

I will be inducing apoptosis in HEK293 cells (transfected with a fluorescent protein) with PMA (0.01uM-10uM) then passing them through a flow cytometer to analyse levels of apoptosis. I plan to trypsinise my cells, spin, resuspend in FACS buffer and to this add the relevant concentration of PMA, incubate for 20-30 minutes and then analyse forward and side scatter by flow cytometry. Does this sound like a suitable protocol? I know that PMA is very potent so are my incubation times too long or my concentrations too high for this purpose? As I don't want to kill my cells before analysis, I want to see a gradual effect by increased PMA concentrations.

Any tips will be really appreciated!

range is okay, you will do inermediate conc (0.1, 1 µM)? incubate at least 1h

Maybe include a low dose (100-200nM) that wouldn't kill 'em for negative control?

QUOTE (The Bearer @ Jul 23 2007, 12:16 PM)

QUOTE (Debz @ Jul 22 2007, 09:57 PM)

I will be inducing apoptosis in HEK293 cells (transfected with a fluorescent protein) with PMA (0.01uM-10uM) then passing them through a flow cytometer to analyse levels of apoptosis. I plan to trypsinise my cells, spin, resuspend in FACS buffer and to this add the relevant concentration of PMA, incubate for 20-30 minutes and then analyse forward and side scatter by flow cytometry. Does this sound like a suitable protocol? I know that PMA is very potent so are my incubation times too long or my concentrations too high for this purpose? As I don't want to kill my cells before analysis, I want to see a gradual effect by increased PMA concentrations.

Any tips will be really appreciated!

range is okay, you will do inermediate conc (0.1, 1 µM)? incubate at least 1h

Many thanks for the replies.

Yes, I plan to do four concentrations of 0.01, 0.1, 1 and 10uM.

I will increase incubation times.