I have followed all the steps shown in the ATCC manual for the HepG2 cell line. Under the microscope, cells look about 80% confluent, yet when I add 2mL to a 75cm flask, incubate them for 4 mins at 37 C, cells are still adhered to the flask. I have tried to pat the flask, yet no results at all. Any help would be appreciated.
Dear Dr Jirau,
my suggestions:
1. Make sure you rinse well with PBS to get rid of any leftover serum in the media before adding the Trypsin. The serum has natural trypsin inhibitors that will deactivate it.
2. Use a new pre warmed trypsin-edta
3. try a higher concentration of trypsin-edta solution.
trypsin 0.25% plus EDTA 0.05% vs 0.025% trypsin 0.01% EDTA
(0.25% trypsin with 1 mM EDTA at 37C for 5 - 10 min)
Best Wishes
As George Sflomos has suggested you have to increase the volume of trypsin. 2ml trypsin is less for 75cm2 flask. You can also increase the concentration of trypsin .
Like other people have suggested
1) Make sure you don't have any leftover serum in the flask before you add trypsin. Increase the incubation time of trypsin upto 10 minutes.
2)Prewarming trypsin upto 37 degrees would be preferred and give your cells good washing with 1X PBS (also prewarmed). Check that your PBS doesn't have calcium/magnesium salts.
3) Alternatively you can wash with 0.5mM (or high concentration) EDTA instead of washing with PBS since you are finding it difficult to detach the cells. Or you can use EGTA instead of EDTA since their effectiveness to strip calcium is higher as compared to EDTA .
4) Use of sterile cell scraper can be employed if the trypsin is not working though it is not suggested to use it too frequently for regular passaging of the cells
5) Check the confluency of the cells as sometimes overconfluent cells display enhanced tight junction connection and protease like trypsin become ineffective under such circumstances.
5) Search literature with regard to whether HepG2 cells produce their own collagen or not. If they do then you should add collagenase as well for detaching the cells.
Thank you all for the help! I applied most of your recommendations and I finally got them to detach. It seems that additional time during trypsinization as required. As well, I increased the concentration of trypsin and left at 37C, 1.2x the normal time of 4-7 mins.
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