PMA activation of cells - (Jul/06/2006 )

Hi all,

I am interested in activation U251 cells with PMA and then check the expression levels of a particular protein using RT-PCR. I went through a few papers to get a protocol and found that cells were grown in complete media till 70% confluence and then cells were grown in a serm free media for 16-18 hrs prior to PMA activation. However I also found a few papers in which they have not used any serum free media and directly activated the cells using PMA.

Now can anyone tell me what can be real use of growing cells in serum free media and also if this step is necessary or not?

Regards,
Anshul

hi Anshul !well use of serum free media depends on your work. since serum contains many growth factors the cells are always in a dynamic state( stimulated state). i.e., they will be responding to external cues. there is a continuous activation and deactivation of secondary messenger molecules.these components might interfere with your cell signaling studies. cell signaling studies are made in serum deprived state (low serum) or serum free conditions.
cheers

hi,
another reason for starvation of cells is to control the growth of cells (to achieve a state where all cells will be in same growth phase).
next time just use one more control where u grow cells in 10% serum, stimulate them with PMA and compare RNA levels for ur protein of interest by RTPCR with results from serum starved cells, if u donot find much difference u can as well skip the starvation step.

gud luk
payeli.

Thanks for the valuable suggestions. I think I will run two sets of experiments - one with the cells grown in the normal serum containing media and second with the cells grown in serum free media. For this I plan to grow the cells in normal media till they reach about 70% confluency and then supplement the normal media with serum free media and keep them to adapt to the new environment for 16-18 hrs. then I treat both sets with PMA for varying time periods.

Does this scheme sound reasonable to you? Apart from this can you also suggest something regarding the concentration of PMA to be used. I read in a few papers where people have used concentrations like 100nM or 50 nM. Also should we make the solution of PMA in water or DMSO or just add the weighted amount directly to the media.

Thanks once again for your valuable correspondence.

Regards,
Anshul

hi,
regarding experimental setup, what ever u mentioned is fine, but include one more set with different concentrations.
here is the experimental setup
-grow cells till 70% confluency
-spin down equal number of cells in different tubes
-suck off medium
-group A-resuspend cells in starvation medium
-group B-resuspend cells in 10%medium
-incubate them for desired time (14-16 hours)
-group A1-unstimulated
A2-50nM stimulated
A3-100nM stimulated
-group B1, B2 and B3 same setup as above.
-proceed with rest of the experiment
decide which ever works fine in ur hands

for the solubility of PMA follow the link
https://www.sigmaaldrich.com/sigma/product%...et/p8139pis.pdf

gud luk
payeli

i think serum starvation of your cells before treatment with PMA would be good idea, since you will see quite strong ERK(MAPK) activation in serum-containing medium.
PS, you could dissolve PMA in DMSO and make as 2mM stock solution, then you could add into medium straight or need to dilute further depend on the concentration you will use.