Clumping of normal colon cells due to transfection reagent - Counting problems (Oct/04/2007 )
I'm using 18Co normal colon cancer cells for siRNA studies using Xtremegene transfection reagent (Roche). Two days after transfection, I'm trying to count the cells on a hemocytometer but they are too clumped. Non-transfected control cells are not clumped. Any ideas on how to reduce the clumping? If I wait for cells to recover from the transfection might the clumping lessen?
Thanks in advance.
How long do you want to wait, and are the FC receptors being bound due to the transfection?
Well, I need to wait until my siRNA has blocked my target, which could be 2-5 days depending on other cells we have used. I have to do westerns to determine if the target was blocked.
Don't know anything about FC receptors....
Could these cells be activated due to transfection?
Do you see cell death? Do you think DNase treatment would help?
If the these cells are dead, then DNA would be present and helping to clump the cells. Maybe when the cells are activated due to the transfection, the FC receptors are causing the adherence. I know of many products to use to keep cells from clumping by blocking the FC receptors. proteins that will bind to the FC receptors will block the aggregation of cells.
I don't see any cell death.
I'm going to count them and then spin down to pellet cells to make samples for westerns.
Somebody suggested collagenase, but how much to I add and when? During or after trypsinization?????