PMA Ionomycin Cell Culture / Dilution and Math Check - (Aug/14/2008 )

Hi-

I am new to cell culture. I am working with a promoter for a gene in Jurkat Cells that needs stimulation by PMA and Ionomycin. Based on a GFP control, I am pretty sure that my transfections are working. Based on my luminometry results, I am pretty sure that I am getting good expression of my SV40 control plasmids. My current problem is that I see no differences in expression between my cells that have been stimulated PMA/Ionomycin and my cells that have not. For the gene I am interested in, expression should be heightened substantially. I am concerned that I am doing something incorrect with the dilution, etc of the PMA and ionomycin. I can not find a clear protocol for how to work with them. Here is what I am doing with the PMA/Iono

My target in the cell culture is Ionomycin 1.5 uM Final concentration:

For this, I got 1mg of Ionomycin calcium salt and I diluted it in 1.35 mL of DMSO. This gives me a 1 mM solution. To a 1mL cell culture I add 1.5 uL of this 1mM solution. This gives a final 1.5uM concentration. I store the stock at 4'. All dilutions done in DMSO.

My target for PMA is 20ng/mL Final concentration:

For this, I ordered 1 mg of PMA and I diluted it in 1 mL of DMSO. This gives me a 1ug/1uL stock. I diluted this 1:10 to give me a 100ng/1uL dilution. I then diluted this 1:10 to give me a 10ng/1uL dilution. I used 2uL of this per 1mL of cell culture. This gives a final concentration of 20ng/mL. I store these PMA stocks at -20'.

I add these both about 2 hours post transfection (nucleofection). I lyse and do luminometry after 24h. I am getting good transfection based on a control pMax GFP plasmid (80% eff). The problem is that there is no difference between the stimulated and unstimulated cells.

Any help would be appreciated!

Mike