Hepg2 cells resistant to trypsin digestion - (Sep/07/2006 )

Hi, everybody:

I am using the Hepg2 cells for the first time. My problem is I couldn´t get them off the flask after 10 minutes with 0.05%Trypsin and I wonder if anybody uses a higher concentration or a longer incubation time or whatever.

Thanx a lot

some cells need to be incubated up to 30 min; better use recombinant trypsin solutions; is EDTA in your solution?

I've been working with Hep G2 for a little while now. I first wash the cells with 1x PBS (this removes any excess serum that can disrupt the activity of the trypsin) and then I use 0.25% trypsin-EDTA. The cells come off rather quickly and I've never had to leave them in the incubator for more than 10 minutes.

try wash your cells once with d-PBS w/o Ca, Mg.

Try trypsin with a higher conc. . this might help.

We have used these cells in the past and yes they can be resistant to trypsin. Try :-

Trypsinising the cells prior to confluence i.e prior to 70% confluence.

Wash with Versene (EDTA) x2 and then x3 with PBS "A" (w/o Ca/Mg).

Try different TC plastic i.e. all cells adhere differently.

I was using HepG2 too. It can be trypsinized easily in my hand. Try to wash it with PBS without Ca/Mg ions. On the other hand, is this trypsin ready-to-use? I found that the ready-to-use with phenol red works more effectively than the one preparing from 10X.

[quote name='alejandrogd' date='Sep 7 2006, 10:29 AM' post='67822']
Hi, everybody:

I am using the Hepg2 cells for the first time. My problem is I couldn´t get them off the flask after 10 minutes with 0.05% and I wonder if anybody uses a higher concentration or a longer incubation time or whatever.

Thanx a lot
[/qu
try with 0.25% Trypsin. then you can get them off the flask easily little than 5min.
good luck!