I am in the process of setting up a series of experiments which will involve incubating U937 with difference cytokines, such as IFN-gamma. I will then do some real-time PCR and possibly ELISA on the supernatant.
I am growing the cells at the moment using the recommended supplements (10% hiFBS, as well as Pen-Strep). The question I have is because I am interested in cytokines in response to my stimulant should I be serum-starving these cells prior to my experiment. As surely the supplement serum will contain cytokines. If a serum starvation is required how long should this be done for or does this vary depending upon the cell type? My worry is that the viability will drop without the hiFBS.
If anyone has any experience with U937 cells then I will be intrigued as to what your experience is with this approach.
Thanks in advance!
Hi Steven, starvation of U937 cells using serum free RPMI 1640 medium for 24h is possible
regards
Cytokine genes may respond to the stimulant rapidly. Thus, you may want to set up a time course experiment, giving different time points such as 2, 4, 8, 12, and 24 hours.
Good luck!
U937 cells are human cell line. If you will use FCS, you don't need any serum starvation procedure for cytokine assay .
Good luck
Agata, do you you measure TNF-a secretion by ELISA or just expression by qPCR? Thank you for your helpful contribution.
Thank you Agata, I have just downloaded your Nanotoxicology paper.
Do you know what the product code for the Sigma PMA was? Did you prepare it in DMSO as suggested and then dilute in culture media (containing 10% FCS)? I assume the cells were at normal culture density (around 5X10^5/ml) when you stimulated with PMA for 24 hrs? I very much appreciate your help.
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