Even though giving tripsin treatment for long time, still i can see clusters while doing cell counting. Please suggest me any tips.
It is best to rinse the layer of cells with Trypsin-EDTA to remove all the serum proteins and then to add fresh Trypsin-EDTA solution. Check cells dispersal under an inverted microscope untill cell layer is well dispersed ( 5 to 15 minutes, may be done at 37°C if cells are difficult to detach).
In order to avoid clumping during dissociation, do not agitate the cells by hitting or shaking the flask while waiting for detachment of the cells. Homogenize the trypsin-cell suspension by pipetting delicately up and down several times, add FBS containing medium to stop trypsinization, pellet down, resuspend cells in the appropriate medium, count cells, adjust cell density and plate in the wells.
I am having the same issue. I was told to pass the cells through a 18g or 20g neddle/10ml luerlock syringe a few times gently after you resuspend the pellet.
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