Before peeling off cells look very healthy. The media also looks pretty clear. So I think no contamination. I am using passage around 20.
Hi Gautam,
It can happens for several reasons: manipulation, higher stimulation or passage number...
Firstly, ageing of 3T3-L1 cells is very important because of their ability to accumulate fat that decreases with increasing number of passages. I will start to use a vial of younger cells and check if they also detach so easily.
After adding insulin post-differentiation medium, there are changes on cell shape, size and cytoskeleton …during this transformation, cells become very sensitive to manipulation. Usually, cells do not detach, but sometimes it can happen. Try to be careful with cell medium exchange.
If the problem persists, you may reduce incubation time with the first differentiation medium for a minimum of two days ... this usually does not compromise differentiation.
Good luck
Adriana Rodrigues
Hi Gautam,
It can happens for several reasons: manipulation, higher stimulation or passage number...
Firstly, ageing of 3T3-L1 cells is very important because of their ability to accumulate fat that decreases with increasing number of passages. I will start to use a vial of younger cells and check if they also detach so easily.
After adding insulin post-differentiation medium, there are changes on cell shape, size and cytoskeleton …during this transformation, cells become very sensitive to manipulation. Usually, cells do not detach, but sometimes it can happen. Try to be careful with cell medium exchange.
If the problem persists, you may reduce incubation time with the first differentiation medium for a minimum of two days ... this usually does not compromise differentiation.
Good luck
Adriana Rodrigues
Thank you Adriana and saber..I will use this time a lower passage number.
Hi Gautam ,
I had the same trouble , my cells detached at day 6 post-differenciation .
What I did, was to handle gently the cells , for example while changing media every 48 hours , I washed the cells only once with PBS instead of 2 times , being careful with the pipeting pressure. The passage of my cells is always arround 15 .
and since then , no problem .
Good luck.
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