Tryspin not working - (Apr/21/2014 )

Hi,

I am a beginner in cell culture world and i was wondering if i could get some help. I have been trying to detach HeLa cells from the bottom of a T-25 flask using 0.5% Trypsin and unfortunately it has not worked. The protocol i use is as follows:

1- Change the flask medium and load with 2.5 ml of 0.5% Trypsin diluted in PBS (1:5).

2- Incubate for 5 min check for detachment of cells. 

3-If enough yield was obtained, add the medium to deactivate trypsin, centrifuge at 1000 rpm for 3 minutes, and re-susupend in fresh medium. 

Unfortunately, when i incubate the HeLa cells with trypsin i do not see any detachment. i have increased the incubation time to 15 minutes but still the yield is so low. I was wondering if i could have your opinion on this.

Thanks 

Be sure to rinse away all traces of serum from your flask before adding trypsin.  Serum deactivates the trypsin so you need to remove all the growth medium, then rinse the cells with 20 mls or more of sterile 1 x PBS, HBSS or media without serum before adding trypsin.   Incubate at 37 degrees for 5 minutes.  Tap flask a few times and your cells should be lifting.  Trypsin with EDTA works better than trypsin alone.

Agree with ddukes post, especiallly mildly tapping the flask on its sides can work wonders !