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HaCaT cells die after trypsinization


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#1 bdtx10

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Posted 11 June 2010 - 07:18 AM

Hey all,

I've been working with HaCaT cells for the past 9 months, and generally splitting them and re-starting cell culture from frozen cells goes fine. Lately though, all of a sudden I can't get them to grow after trypsinization. I am trypsinizing with 0.05% trypsin-EDTA from GIBCO for 15 minutes at 37C. Generally this works, but when I trypsinize cells that seemed to be growing fine from liquid N2, they don't reattach and soon die. I am thinking general trypsinization may be too harsh for cells that have just been frozen? Many of my frozen stocked cells hadn't gone through too many passages, so I think I just need to find a way to make trypsinization safer for them.

I've seen that some people treat cells with EDTA in PBS at 37C first. Does anyone recommend doing this first for situations where treating cells just with trypsin-EDTA is too harsh, like for new cell cultures?

thanks!!

#2 Inmost sun

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Posted 12 June 2010 - 10:31 AM

Hey all,

I've been working with HaCaT cells for the past 9 months, and generally splitting them and re-starting cell culture from frozen cells goes fine. Lately though, all of a sudden I can't get them to grow after trypsinization. I am trypsinizing with 0.05% trypsin-EDTA from GIBCO for 15 minutes at 37C. Generally this works, but when I trypsinize cells that seemed to be growing fine from liquid N2, they don't reattach and soon die. I am thinking general trypsinization may be too harsh for cells that have just been frozen? Many of my frozen stocked cells hadn't gone through too many passages, so I think I just need to find a way to make trypsinization safer for them.

I've seen that some people treat cells with EDTA in PBS at 37C first. Does anyone recommend doing this first for situations where treating cells just with trypsin-EDTA is too harsh, like for new cell cultures?

thanks!!


use a less aggressive trypsin solution, f.e. TrypLE from Invitrogen;

indeed, some detach their cells by only treatment with EDTA




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