I'm working on IGF-1 effects on cytokine expression in CD4+ cells. According to BioLegend reference, I used 500ng/ml ionomycin and 500ng/ml PMA to stimulate splenocytes as well as GolgiStop (BD product). Medium I used RPMI 1640 with 10% FBS. While 4 hours later I noticed that most of the splenocytes die no matter with or without IGF-1 treatment. Is that I added to much PMA? I noticed in publication, PMA concentration is from 5-500ng/ml. I want to check IL-17 and IFN-gamma. what concentration would be reasonable for my experiment?
Also, for GolgiStop, I can add with PMA and Ionomycin or add one hour before stimulation stop?
Hey Tong,
I'm also working with CD4+ TC and when I started doing the whole stimulation stuff I checked various sources and now I'm successfully using:
In RPMI 1640 +10% FBS: PMA 50 ng/ml; Iono 500 ng/ml and after the first hour I'm adding Brefeldin A to 5 µg/ml and/or Monensin to 5 µg/ml (depending on the cytokines of interest), which equals the GolgiStop/Plug. These are all the final concentrations in the medium. Setting up aliquots of 10x stocks of these makes your life a lot easier.
Some cytokines are processed to maturity 'later' in the cellular synthesis pipeline than others, which is why you could run into problems when adding GolgiStop/Plug right from the start. But there are tables for that kind of information. Perhaps check on the BD homepage etc, they have pretty useful resources for that.
For IL-17A and IFNy detection and others, I stimulate with PMA/iono for an hour and then add BFA + Mon for another 3 hours. Works like a charm.
To my knowledge, BFA and Monensin start becoming toxic for the cells 'at some point', so one should not massively exceed the incubation time.
Hope this helps. Good luck!
Hi Tong.
I have done more in vitro assays than I care to remember (mostly looking for IFN-g), and I have always used 1ug/mL of both Ionomycin and PMA (final concentration, though PMA can be halved...more a matter of convenience in diluting for me), generally as my positive control, as we also use plate bound antibodies for targeted stimulation. I do 4 hours total incubation, 1 million cells per well in a 12-well plate (RPMI-10%), adding brefeldin (Golgi Plug, not Golgi Stop) after 1 hour, very much like Eric. Brefeldin is not quite so toxic as monensin, and I have actually incubated cells for longer than 6 hours with little detriment.
I suspect the problem might be something else. Speaking from experience (and plenty of failures), the processing of the tissue and the time it takes to get the cells in warm media is vitally important, especially with splenocytes, or any tissue bound cells, really. I've found that, the faster I'm able to get cells into warm media, the better my outcome. You are likely losing a fair number of cells if it takes longer than about an hour and 20 minutes to go from mouse to culture. I've also done this assay with PBMCs, and almost always have success...likely because it tends to be much faster to isolate cells from blood than tissue.
I hope this helps. Take care!
You're perfecty welcome, Tong.
I'm working with cell numbers/densities way below that level so Lucas can probably help you better with that question.
Dear Tong,
in addition to the useful answers above:
I have used only 10 ng/ml PMA in all my assays and it is enough for maximal stimulation. I guess using 500 ng/ml is very likely to be responsible for the massive cell death you observed. I used 1 µg/ml Ionomycin just as Lucas and added BrefA after 1h for another 3h at 5 µg/ml.
Beware that depending on the cell source, RPMI might not contain sufficient Ca2+ to saturate the ionomycin response so you could use IMDM instead (http://onlinelibrary.wiley.com/doi/10.1002/eji.201445247/abstract).
I have also titrated the cell density and there was a significant reduction in the frequency of cytokine-expressing cells between 2x10e6 /ml and 1x10e7/ml, so I wouldn't recommend to go a lot higher than 1x10e6 cells / ml.
Good luck,
Jakob
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