4 replies to this topic

[Annik]

Hi everybody!

I am culturing HepG2 cells and I would like to know what are the better conditions to trypsinise the cells. I have been use 5 mL of trypsin for 5 min in the incubator, without washing the cells with PBS. The cells detach quite well but then they form horrible clumps and now they look like if they have some vacuoles or something appearing on the top of the them. Could anybody give me some tips in order to get a better culture?

Thanks in advances,

Annik

[zienpiggie]

I find that when I subculture before they get too confluent, they won't form clumps. The HepG2 was indeed finicky and prone to forming clumps.

[bob1]

Also try using a shorter time in trypsin - over treatment with trypsin leads to clumping.

[Annik]

Thanks a lot!! Actually I used trypsine for a shorter time and now the culture looks better!!

[bob1]

Good.  You only ever need to trypsinise until the cells are detaching (observe them under the microscope, or just by eye when you are more experienced) - using a defined time is too restrictive and can cause a lot of damage to the cells.