GH3 cells - (May/10/2012 )
Currently I'm working on GH3 cell lines. But contamination always happen. When I check the media, the media is okay.
When I'm doin the subculturing, I always make sure I'm doing it aseptically. Lets say if I'm not sure whether the pippete had touch something in the laminar flow/clean bench, I will change to a new one.
I did notice this GH3 cell very easy to get contaminated compared to HepG2 cells.
Please give some advice...
Thank you in advance.
Do you put antibiotics in your media? If so, have you checked for low level contamination in your stock cells as the antibiotics may hide the low level contamination for quite some time?
Have you checked your trypsin and PBS (or whatever you use to wash your cells before subculturing) for contamination?
Thanks Kat T`~~~
I never check the trypsin. And for PBS, I'm really sure its not contaminated cos I use the same PBS to subculture HepG2.
I put antibiotic in my media but i never check the low level contamination of my stock cells, may you explain how to do this?
Sure, I make up some sterile nutrient broth (you'll only need about 100mL). You'll also need two sterile 125mL erhlenmeyer flasks (to sterilise the flask autoclave it with a plug of cotton wool topped with tin foil)
Using aseptic techinique:
Add 50mL of sterile nutrient broth to each of your sterile flasks.
Take one of your frozen cell stocks and thaw it, add it to one of the flasks, and label the other flask as control (or blank)
Grow your flasks at 37°C in a shaking incubator for about 3 days.
This is how I test all of my tissue culture reagents when I have a contamination issue. So you could make up more of the nutrient broth and sterilise more flasks if you wanted to check your media, trypsin or any other reagent. Also, if you felt that you didn't want to waste the cells, you could always spin down the cells and remove the supernatant and test that, reconsititue the cells in media and grow them as per usual. The contaminants will be in the supernatant.
Thanks Kat T~~
This is very informative and I will try~~