Avoiding cell clumps - (Mar/28/2007 )
Hi guys,
I'm working with an adherent pituitary cell line (AtT20) and I'm performing an MTT test to determine the efficacy that several drugs have on the viability of such cells. The problem I have is that after the trypsinization the cells tend to cluster and as I centrifuge the cells after this step this tendency tend to be even stronger.
Before plating cells in microtitration plates I flick and pipet cells vigorously to avoid clumps. Then I plate them and after a four days time course I see differences among the three wells similarly treated (that means same amount of drug and same exposition time). This lead to different problems:
1) I have a large standard deviation for each conditions
2) but particularly I'm not confident in the cells behaviour after drug administration.
Has somebody of you had the same problem and how did you fix it?
Thanks
well, did u try to use EDTA?
as a chelating agent, EDTA removes (chelates) cations (like calcium) from cell surfaces which are responsible for cell-cell adhesion...
check this topic
also, trypsinization time and concentration differes from one cell type to another..
and try to apply mild centrifugation...
I'm working with an adherent pituitary cell line (AtT20) and I'm performing an MTT test to determine the efficacy that several drugs have on the viability of such cells. The problem I have is that after the trypsinization the cells tend to cluster and as I centrifuge the cells after this step this tendency tend to be even stronger.
Before plating cells in microtitration plates I flick and pipet cells vigorously to avoid clumps. Then I plate them and after a four days time course I see differences among the three wells similarly treated (that means same amount of drug and same exposition time). This lead to different problems:
1) I have a large standard deviation for each conditions
2) but particularly I'm not confident in the cells behaviour after drug administration.
Has somebody of you had the same problem and how did you fix it?
Thanks
Dear eyes,
I have had this problem in the past with some cell lines. The question I have for you is WHEN do you do the trypsinisations? i.e at 60,70,80,90,100% confluence. Try trypsinising at different confluency levels. What I found was that if I left the cells to get to full confluency, then the cells would come off the flask as normal but then immmediately aggregate into clumps. Once in those clumps it was impossible to get a single cell suspension, which is what everybody tries to achieve when seeding for experiments. I have had to compromise with the maximum number of cells obtained from a flask.
Nothing tried, nothing gained as my mum would say.
Regards
Rhombus
Thank you Rhombus for the information.
First, thanks to every body
For strawberry: I'm using a 0.25% Trypsin w/o EDTA, so probably I could try to see what will happen using it.
For Rhombus: the cell line I'm using do not become 100% confluent, so normally I trypsinize at 80%. Probably first I will try using Trypsin/EDTA and then changing the moment of trypsinization.
Do you know if clumping could be consequence of shaking of flasks during the trypsinization?
I have had this problem in the past with some cell lines. The question I have for you is WHEN do you do the trypsinisations? i.e at 60,70,80,90,100% confluence. Try trypsinising at different confluency levels. What I found was that if I left the cells to get to full confluency, then the cells would come off the flask as normal but then immmediately aggregate into clumps. Once in those clumps it was impossible to get a single cell suspension, which is what everybody tries to achieve when seeding for experiments. I have had to compromise with the maximum number of cells obtained from a flask.
agree with u Rhombus, it's a good suggestion
Thanks guys,
I probably solved the problem using Trypsin with EDTA 0.53 mM
Eyes