PMA preparation and stimulation
I bought the PMA (1mg) from sigma, and I am ready dissolved it by adding 1ml DMSO (stock solution 1 mg/ml). Then the PMA solution will be divided into 100 tubes and store them at -20℃.
I want to use the PMA solution to stimulate THP-1(1x106 cells/ml), so I will choose one tube of PMA and dilute it by adding 990uL PBS so that the concentration of PMA reach to 10ug/ml. 25uL PMA solution will be added into 5mL THP-1 medium for stimulating (PMA concentration is 50ng/ml). Last, I will treat the cells with LPS (0ng/ml, 10ng/ml, 100ng/ml and 1000ng/ml) and test the cytokine levels (IL-8, IL-6, TNF, IL-12p70, IL-10 and IL-1B) at different time points.
I want to ask: 1) whether the DMSO need to use 20mM? I have the DMSO bought from Sigma(D2650, ≥99.7%)
2) How many hours will take for PMA stimulation? Or whether I need to change higher concentration of PMA?
Dear Liu, kindly follow the instructions given by Dr Anderson. We too have used PMA at 5ng/ml for 5h and found it to be sufficient for inducing monocyte to macrophage differentiation. Following differentiation, you may replace PMA media with complete growth medium. Allow cells to attain confluency and incubate overnight in basal medium before challenge with LPS (50-100ng/ml).
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