What 's the minimum time frame (in days/hours) required for serum starvation in NIH3T3 cells to induce quiescence?
It is critical that you grow the cells to confluence first, or else you will not get a good serum starvation response. Most people use 0.1% FBS for 48 hours as Neus suggested. However, the mitogenic potential of every lot of serum is different, so for some people, 1% works fine whereas others find that even lower serum concentrations are needed.
48 hours sounds about right. Its pretty quick. We did it in 1% FBS (usually use 10% FBS in DMEM and senescence occurred quickly- see Bosse et al. Cancer Research 2012 (Figure 6 C).
Hi Praveen,
In NIH3T3, 1% FBS for 48 hours leads at least 25% less viable cells, however it increases the percentage of cell in GO/G1 phase in 95-98%.
It is critical that you grow the cells to confluence first, or else you will not get a good serum starvation response. Most people use 0.1% FBS for 48 hours as Neus suggested. However, the mitogenic potential of every lot of serum is different, so for some people, 1% works fine whereas others find that even lower serum concentrations are needed.
Thank you Fabiola Filippin Monteiro & all for the inputs. I am growing them to confluent in 10% FBS now. Let me try it for 48 hours then in less than 1% serum medium.
HeLa cells can survive without serum (only DMEM) for one week. During this time cells divide. I do not split cells, just change fresh DMEM without serum. I don't know, but I think it should work similar way for NIH3T3 cells. 48h time frame looks enough, but it all depends what is your experiment. If you are looking for cell cycle related flow experiments, more time without serum is better. If you are doing signaling experiments, overnight to 48h without serum should work.
No, Hela is not comparable to NIH3T3, and can survive all manner of insult that other cell lines won't tolerate. NIH3T3s will die when cultured in low serum or serum-free medium for extended periods of time. While some cell death can be tolerated in this assay, you will not be able to extend the starvation period beyond 96hrs without major losses in cell viability. If you can't get your 3T3 cells to synchronize in 48hrs, I'd get earlier passage cells or an entirely new lot from ATCC, rather than extend the starvation period to abnormally long intervals.
By the way, why not starve them without FBS at all? What will be the downside?
I agree with Fabiola fibroblast cell viability is lost in a large proportion of cells at 1% FCS. I usually wean my cells off it by a reduction of 1% each day.
- Badges
- Science topic