HEK293 trypsinisation - (Feb/16/2007 )
Hi,
The day following passage of my HEK293 cells they look quite sickly and do not seem to adhere nicely to the flask surface and there are lots of floating cells too. Could this be due to the trypsin or is it just because the cells are only semi-adherent? Is there a gentler way to passage this cell type that might promote better re-adherence of the cells?
Advice would be greatly appreciated!
The day following passage of my HEK293 cells they look quite sickly and do not seem to adhere nicely to the flask surface and there are lots of floating cells too. Could this be due to the trypsin or is it just because the cells are only semi-adherent? Is there a gentler way to passage this cell type that might promote better re-adherence of the cells?
Advice would be greatly appreciated!
after one day of trypsination cell should have recoverd; it depends on incubation time and type of trypsin; use better recombinant trypsin/EDTA solution (f.i. TrypLE), and follow the recommended incubation time for your type of cells; consider that trypsin does not only lyse attachment proteins but also extracellular domains of growth factor receptors which is harmful to cells
the old hek293 are generally quite robust. i use trypsin edta as well - but you shouldn't need to leave it on for too long. what volume did you use?
how did they look before you split them?
any chance of contamination?
also - straightforward thing but happens to the best of us - if your lids aren't vented did you loosen the cap?
how did they look before you split them?
any chance of contamination?
also - straightforward thing but happens to the best of us - if your lids aren't vented did you loosen the cap?
i use a cell dissociation fluid (non enzymatic) from sigma and the cells fall of in a matter of minutes and look fine after passage
how did they look before you split them?
any chance of contamination?
also - straightforward thing but happens to the best of us - if your lids aren't vented did you loosen the cap?
I used versene/trypisin (1:1). I usually use about 2 mls to trypsinise a T75 flask and I only leave it on for about 1-2 minutes because they do dissociate very quickly. It doesnt look like contamination and I use vented lids so I usually make sure they are tight. Should I try coated culture flasks (Poly-Lysine? Collagen?)?
Dear Oaktree,
HEK293 cells are not very adherent at all. This is something you'll have to live with when working with them as adherent culture (you can adapt them to fluid culture). They are also one of the few cell lines that can be detached by using EDTA alone and I would recommend doing that instead of trypsin.
You can try and use poly-lysine (you will need to if you want to grow them on coverslides) but usually very careful treatment (do not pipet onto the cells but tilt the flask/plate while pipetting) is enough to retain most of them.
Hope that helps
LeserattePD
how did they look before you split them?
any chance of contamination?
also - straightforward thing but happens to the best of us - if your lids aren't vented did you loosen the cap?
I used versene/trypisin (1:1). I usually use about 2 mls to trypsinise a T75 flask and I only leave it on for about 1-2 minutes because they do dissociate very quickly. It doesnt look like contamination and I use vented lids so I usually make sure they are tight. Should I try coated culture flasks (Poly-Lysine? Collagen?)?
How old are the cells. If they are high passage number , this can happen.