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.