Hello everyone,
This is my first time working with a neural stem cell line. The cells reached me in passage 1. I use PLO and laminin-coated plates for culturing. At the first passage, cells did not deattach even after about 20 minutes with accutase.
My enzyme is brand new and aliquoted but I think there is a problem with the lot. Or maybe I did not wash enough with PBS? Since I do not know much about the character of cells, I try to be sensitive.
Can I use TrypLE instead of accutase? Any information would be very helpful about these cells and culture process.
Thank you.
Hello Gülşah Torkay
Accutase is an enzyme preparation that is proposed to be less aggressive than trypsin. It is formulated at a much lower concentration making it less toxic and gentler on cells, but very much effective. Accutase should be used in situations whenever gentle and efficient detachment of adherent cell line is needed. It works extremely well with neuronal stem cells.
Accutase would be the right choice for neuronal stem cells. But since it is not working in your case for whatever reason, you may use TrypLE instead of accutase. But you need to know that the enzyme concentration and treatment duration are important factors, because of the ability of the enzyme to break down cell surface proteins. For this reason, you may have to dilute TrypLE by 50% (by half the typical concentration), and the duration of treatment must also be well defined say 5 mins as the minimum time.
If you can get hold of another fresh vial of accutase, it would always be better to use accutase rather than trying TrypLE or Trypsin/EDTA on neural stem cell line.
Best.
You can wash the cells twice with PBS (calcium and magnesium free). I can't guarantee this will work, but you can try using the Versene solution to deattach the cells.
Try a few PBS -/- rinses as suggested above (pre-warm your PBS) and also pre-warm your accutase just before you need it. 20 minutes seems very long. For human neural stem cells, shouldnt take even a quarter of that time.
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