I have a question regarding culturing fibroblast IMR90. I use DMEM+10%fbs+1% pen/strep for culturing them. I use 0.05% trypsin EDTA for trypsinization for 40 sec in the incubator and add DMEM + fbs+ pen/strep to neutralize the trypsin. However, I do not get a good viability how early I try to retrieve them from trypsin treatment. It is always between 50-70 % and never in 90s. Also trying to spike them with TGF beta to produce alpha SMA However, I do not get proper staining. Is it because the viability is compromised that I do not get good staining?
Hello Sanchita Mallick
Do you trypsinize IMR90 cells when they are over confluent? During sub-culture, the cells must be 70-80% confluent. Cell death can happen at high confluency because nutrients in the media become depleted or cells start competing for space on the culture dish or flask surface. Therefore, when you harvest cells at high confluency you will get reduced viability.
For trypsinization of IMR90cells, 0.25% (w/v) Trypsin-0.03% EDTA is used, and the cells are exposed to trypsin for 2-4 mins. How do all your cells detach from the substratum in 40 secs? This means that your cells are not in good state of health.
Also, regarding the growth media, you may supplement the media with 2mM L-glutamine, 1mM sodium pyruvate and (1X) non-essential amino acids. Please note that whenever you use cells for any cell-based assays, the cells must be healthy (viability>95%) so that you obtain good reliable results.
Best.
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