Hi there,
I am doing a qPCR experiment and I am going to compare cytokine stimulated cells vs. unstimulated cells' gene expression levels.
I need around 10 million cells (1 X 10^7) for RNA extraction using the RNeasy mini kit from QIAGEN.
I was wondering if stimulating the cells in T75 flask would be better compared to stimulating them in 10mm dish? I worry that 10 million cells would be too crowded in 10mm dish and the cells won't have enough contact with the cytokine.
Hi Min: We only use 6-well plates for growing HK2 cells in the absence or presence of cytokines such as IL1, IL6, TNFalpha, BMP2, and TGFbeta1 by qPCR and Western blotting. Both methods were "home-made" and very sensitive and inexpensive, so that we demonstrated for the first time that kidney proximal epithelial cells actively synthesize plasma proteins and secrete into the apical (lumenal）side in normal situation, but reversed course when cultured under the influence of cytokines. Check out and download our paper in RG for technical details. Good luck.
Hi Min! I will have to concur with Ke-Wei on this. I did my cytokine stimulation in triplicate in 6 well plates for U937 and THP-1. There was plenty of space for not only the population but also for insuring that the cells have "equal" exposure to the cytokine, without wasting media or cytokine. T75, while not the largest, may result in the waste of materials unnecessarily, i.e. the cytokine, by having to dose in that large of a volume. Hope this helps and best wishes!
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