Problems when harvesting cells - (Jul/14/2008 )

I am currently growing HUVEC cells and have encountered problems in harvesting these cells that are already confluent. After i centrifuge the cells, no pellets form.

I use 1 ml accutase and 3 ml PBS and incubate it for 5-10 mins to detach the cells, after which i transfer them int0 2 centrifuge tubes 2 ml each (one vial to cryopreserve and one to subcultivate) at 2000rpm for 5 mins. But after centrifuging no pallets form. and i have tried centrifuging 3 times repeatedly. I am sure the cells were properly detached when viewed under the microscope but how come no pellets were formed?

Can some one help me? Can i still salvage the cells by other means to re-culture it again?

Hi there,

I use HBSS without Calcium and Magnesium (from Lonza) to wash the plates 3 times before I add the trypsin or the non-enzymatic cell dissociation soln.
I use Trypsin versene (hyclone) to detach the cells, but when I need to do protein expression studie, i use non-enzymatic cell dissociation solution from sigma. For a 100mm flask I use 3ml of non-enzymatic solution and keep it for 5-8min at 37 deg.


Another thing with the non-enzymatic solution is that you may need to pippette the solution after the incubation to make sure that you get most of the cells from the plate.


If you want to passage the cells trypsin versene works fine also. For this I use 2 ml trypsin-versene and keep the plate in the incubator for 3 min and then tap the plates with my palm to detach.

Also while spinning i do not go above 1300rpm and I spin the tubes for 8 min.

Let me know if this works for you.


Good Luck

Pooja

Thanks for the tip Pooja I'll give those a try. But i'm still baffled that no pallets form. Where could have all the cells gone? At least it should have a tiny piece formed rite?? Could it be that the cells are very small that they do not form pellets? Or perhaps too little in number that the pellet cannot form?

Thanks for the tip Pooja I'll give those a try. But i'm still baffled that no pallets form. Where could have all the cells gone? At least it should have a tiny piece formed rite?? Could it be that the cells are very small that they do not form pellets? Or perhaps too little in number that the pellet cannot form?

Hey Pronana,

Trypsin Versene works very well, so give that a shot!
and don't forget to wash the cell monolayer with HBSS (without calcium and magnesium) three times.

Also try centrifuging at 1300rpm for 8 min

Pooja