I do inflammatory cell signalling related experiments. I seed A549 cell in a 6-well plate (for eg.) and wait for 48 hours to get them confluent. Then serum starvation overnight, followed by required treatments with inflammatory mediators or drugs etc. Is it better to make the serum starve just 24 hours after seeding on to the plate instead of 48 hours?
If you are serum starving "over night" I would assume that means for approximately 12 hours, not 24 hours. In any case, this all depends on the end point you are measuring. For example, if you will be lysing the cells for western blot analysis, you want to make sure you have a suitable protein concentration for your lysate. Or if you want to measure RNA, you have to make sure you have enough cells where you can isolate enough RNA. If I were you, I would use trypan blue to determine % cell death after 12 hr, 24 hr, 48 hr serum starvation. Then also consider the toxic effects of your treatment (% cell death after 12, 24, 48 hr starvation + drug). If the cells are too stressed (starvation +drug), then you will have confounding factors in your analysis, i.e. the cells are undergoing apoptosis so any changes you see in RNA/protein can be due to apoptosis and not your inflammatory signal. You can do a toxicity assay to determine this (there are kits). You should choose the condition that gives you the response with least amount of toxicity.
hello,
After seeding of A549 cells, let them attach for 24 hrs. After this starve the cells for 12-16 hrs.then you can give treatment.
best of luck
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