LPS is shown to induce SREBP expression in the literature, however, there is inconsistency in the literature methods that states to perform the experiments in serum free media and other papers have cultured in 10% serum containing media. When I culture in serum containing media I have seen variability in my results lately. What would be the advantages of cultured in SFM when assessing LPS induction of SREBPs via qRT PCR?
There is a good chance that SREBP expression (are you looking at 1a, 1c, or 2?) would be sensitive to things in serum, especially insulin. Although I don't know what role this would play in macrophage, but I would still suggest serum-free media.
Also, you could evaluate SREBP expression/activity by measuring expression of its target genes (ACC, FAS for SREBP-1c, HMGCR, LDLR for SREBP-2).
Thank you! I agree with you the serum could be causing inconsistencies. I will serum starve my cells from now on. I have seen that my receptor of interest down regulates both SREBP1 and SREBP2 expression in macrophages. These transcription factors play a key role in inducing foam cell formation in macrophages under inflammatory conditions. For examining SREBP activity, I will be measuring the expression of the downstream genes; however, I was trying to optimize my experiment conditions prior to evaluating any downstream genes. SREBPs after translocation into the nucleus can induce a feed forward mechanism which can be detected by qRT PCR which is why I was only assessing these two genes at the moment. Thanks again for your advise. I really appreciate it!
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