FACS-cells clumbing together - (Apr/28/2008 )
Hi,
I'm new to cell cytmetry. I was identifying CD4 positive cells from huh7 cell lines but after trpsinization, my cells were clumped and was very difficult to resuspend them. Consequently I lost many cells and got a very low peak. Can anyone give me a few pointers to prevent cell clumping, e.g. during cell staining and/or during trypsinization of cells.
Any help would be uch appreciated.
SF
-Sarwat-
QUOTE (Sarwat @ Apr 28 2008, 04:34 PM)
Hi,
I'm new to cell cytmetry. I was identifying CD4 positive cells from huh7 cell lines but after trpsinization, my cells were clumped and was very difficult to resuspend them. Consequently I lost many cells and got a very low peak. Can anyone give me a few pointers to prevent cell clumping, e.g. during cell staining and/or during trypsinization of cells.
Any help would be uch appreciated.
SF
I'm new to cell cytmetry. I was identifying CD4 positive cells from huh7 cell lines but after trpsinization, my cells were clumped and was very difficult to resuspend them. Consequently I lost many cells and got a very low peak. Can anyone give me a few pointers to prevent cell clumping, e.g. during cell staining and/or during trypsinization of cells.
Any help would be uch appreciated.
SF
I've read that the addition of 5mM EDTA to sample prevents cells from clumping. So, do I add EDTA to the cell suspension in PBS buffer after tripsinzation or to PBS before tripsinization?
Also, at what step in tripsinization do I add DNAse to 10 U/ml. Do I add this after I have my target number of cells in suspension?
Thanks
SF
-Sarwat-
Add EDTA with the trypsin, and make sure that you are not over trypsinising cells as this will make them clump together, 1-3 minutes in trypsin is fine for most cells. You have to make sure that you have a single cell suspension before fixing your cells, otherwise you won't get good facs results.
-bob1-