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How to count clumpy epithelial cells? - (Feb/01/2014 )

I have some human epithelial cancer cell lines that tend to clump after trypsinization. The clumps are usually made up of 4-8 cells and this makes the counting difficult and determining how many cells to seed even more confusing. To trypsinize my  cells,  I wash my cells once with 1X PBS, add trypsin then place at 37 for 5-10 minutes. The cells start to release in clumps without tapping. I add complete media to mix and break up the cells as much as possible. During cell counting, a majority of these cells are in clumps in the hemocytometer.

 

Should I try to count all the cells in the clump as individual cells or treat the clump as one cell? I.e. clump of 4 cells as one cell or clump of 4 cells as 4 cells? When I seed the cells, the clump will settle as one cell which is why I thought maybe counting the clump of 4 cells as one cell. 

 

Are some cells just prone to clumping? I have a couple a few lines that do this and the rest do not. 

 

Any method or technique to overcome clumping? Reagent to use, needle and syringe break up clumps, different method of trypsinization?  Please help! 

 

Thanks!

-Epigeneticist-

Some cell lines are particularly prone to clumping, there isn't much you can do about it other than ensuring that you leave the trypsin on for as short a time as possible.  You may be able to prevent some of the clumping by making sure that you use Mg and Ca free PBS and adding about 0.04 mM EDTA to your trypsin.

-bob1-

Some cell lines are particularly prone to clumping, there isn't much you can do about it other than ensuring that you leave the trypsin on for as short a time as possible.  You may be able to prevent some of the clumping by making sure that you use Mg and Ca free PBS and adding about 0.04 mM EDTA to your trypsin.

 

How would you count the cells when they clump, if the clumping is on average 4-8 cells? Count clumped cells as one cell or try to count all the cells in the clump?

-Epigeneticist-

I would count them as individual cells - as when they attach they will most likely form a small colony that will take up the area of the number of cells in the clump.

-bob1-

After trypsin deactivation I centrifuge the cell suspension and resuspend the pellet (in about 1-2ml culture medium) using an 1ml syringe and a 21G needle (gently, no foam). Try 4-6 times at first. When I use ΗepG2 cells, which clump A LOT!, I pass them through the syringe up to 20-25 times. 
 I always get a single-cell suspension and viable cells.

-cell slave-

Repeat up and down several time 

-Mohamed 1984-