I want to isolate single cell clones of HepG2 from the pool using limiting dilution cloning (LDC) in 96-well plates. But I found that it's hard to get single cell suspension of HepG2 (at least 30% cells form clumps) even if I extend the trypsin incubation time (~15min, 0.5% trypsin-EDTA) and pipette the cell suspension up and down after trypsin. What can I do to solve this problem? Thanks!
Thanks for your answer. I used P1000 with a 10 µL tip on the end to pipette the cell suspension up and down. May I ask the type of syringe you used to make suspension? @Ishita Virmani
Hi Mark Ian Fowler. Thank you for your answer. I followed your suggestion and used Accutase to detach the cells. It is indeed better than 0.25% trypsin. However, I still found some cell clumping at a frequency of approximately 10-20%. Could you please provide me with your protocol for breaking down HepG2 cells into a single-cell suspension?
Make sure you pipette them up and down using a 10ml stripette at least 10 times to separate them.
I second the Accutase suggestion, they also have a new GMP grade product that is remarkably good at creating single-cell suspensions with high viability.
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