HOW DO YOU STOP CELLS FROM CLUMPING TOGETHER - (Oct/18/2006 )
I am working with BT20 and for some reason every time I trypsinize the cells they clump up into a big glob once they come in contact with one another and I can't seem to get them back into even suspension. What could cause this and how can I rectify it? 
how long do u leave the cells with trypsin. It seems like nucleic acids r being released from the cells leading to the clumping nature. probaby many cells r dying during this period.
try to reduce the time with trypsin. Dnase might help, but its an unnecessary step if u could avoid clumping by some other way.
how long do u leave the cells with trypsin. It seems like nucleic acids r being released from the cells leading to the clumping nature. probaby many cells r dying during this period.
try to reduce the time with trypsin. Dnase might help, but its an unnecessary step if u could avoid clumping by some other way.
[/quote]
Well generally I leave the trypsin on for about 10 minutes. If I seed the clumped cells, they still adhere and grow, so I don't know if the cells are dying during trypsinizing or not however, I am curious to know how to go about breaking up the clumps to get a single cell suspension again....
i usually leave the trypsin for 3-5 min at 37C and then add media to stop trypsin and pipette it up and down till they r seperated into individual cells. This depends on the cell type. Some can b seperated very quickly while some take a while.
I once met the similar situation. I trypsinized my 293 cells for about 2minutes, then, they clumped... It took me weeks to recover these cells.. It seems a very short trypsinization works better..
Do your cells (clumps) spread on plate? In my case, they attach, but not spread.. (I change to a new dish and trypsin briefly now)
I am not sure of your cell type but for monocytes about 3 mins with Trypsin is adequate.
The best thing to do is be careful and monitor it under microscope.
What we do is after 3 mins of incubation with trypsin, we check under microscope. When the cells get detached, they get rounded and then U may harvest them. If they have not, we keep for a few minutes more and check again.
Do your cells (clumps) spread on plate? In my case, they attach, but not spread.. (I change to a new dish and trypsin briefly now)
Thanks for your responses everyone! My cell line must be extremely sensitive. Yeping: yeah I've had a horrible time trying to recover my cells, the clumps do attach as you've said and hardly do they spread...cells do branch out around the perimeter of the clump but they don't grow normally at all.
use trypsin-EDTA, it prevents clumping.....or so ive been told
and it states in the ATCC data sheet to use trypsin EDTA too . . . . .
Protocol:
Remove and discard culture medium.
Briefly rinse the cell layer with 0.25% (w/v) Trypsin- 0.53 mM EDTA solution to remove all traces of serum that contains trypsin inhibitor.
Add 2.0 to 3.0 ml of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting.
Add appropriate aliquots of the cell suspension to new culture vessels.
Incubate cultures at 37°C.
(ATCC website)
We use PBS to rinse. I wonder if there is any advantage of rinsing by Trypsin-EDTA over PBS