they come up in clumps - (Jul/01/2007 )
I am now working with ht29. The cells are growing well and and i have been subculturing them. However, I have been trypsinizing them for 6 minutes, but they don't come up. So i hit them gently, then they come up in clumps. I tried to pipette up and down for 12 times, the clumps remain. This will affect my result of MTT assay later. How can i make them into single cells solution?
cells tend to stick together due to membrane protein adhesion, try to use protease if possible...
have you tried using EDTA in your trypsin?? I have a very sticky cell line also, and I found that adding EDTA to my trypsin helps lift them faster, as well as fewer clumps which are not too difficult to disperse by pipetting a few times.
I support this idea: Cell-cell adhesion is organized, among others, by cadherins which is a Ca2+-dependent process...; so try EDTA along with trypsin; moreover, you may incubate longer...
my trypsin contains the EDTA as well. So maybe i need to increase the incubation time. However, i afraid that exposure to trypsin for too long a time will affect my cells viability. 6 minutes is according to a protocol. so maximum i can incubate for how long? anyone with experience in this?
I used to have a similar problem with SHSY5Y cells. I would trypisinze them for 5-6 min. and then tritrate them for 5-6 min. Only after vigorous titration, I would get single cells. Else there is always some remaining clumps.
Good Luck !!!