I am currently working on a study using RAW 264.7 cells. I am polarizing the cells to an M1 phenotype. I am then applying an anti-inflammatory compound 24 hours later. These cultures are being maintained for 5 days, and I am collecting media samples for protein analysis at 1, 3, and 5 days after the addition of our anti-inflammatory compound. However, I am concerned that the acidity of the culture media is affecting my results.
Has anyone cultured RAW 264.7 cells for protein analysis over several days (past 48 hours)? If so, how often did you perform media changes? Would frequent media changes (i.e. days 2 and 4) lead to a diluted amount of proteins in the media?
Thank you in advance
Hi Samantha Hall
RAW 264.7 cells, a type of macrophage, are known for their morphological plasticity. This means they can readily change shape in response to their environment.
I assume you are giving the drug treatment in plain media (without FBS), that could be the reason the media is getting acidic. I would recommend you to use at-least minimum FBS media (2% so as to limit the stress on cells). Further, you can culture the cells in more number of dishes and can collect the medium from different dishes on day 1, 3 and 5.
Thanks,
Samir
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