When I recover the PK-15 cells from the Liquid nitrogen after 48h I passage it, I use 1.5mL trypsin for 5 minutes at 37C to detach them. They detach very well but when I passage it on 2nd, 3rd and so on passages I use the same amount of trypsin and incubation time but the cells are unable to detach.
please give me some suggestions to solve this problem.
thank you in advance.
If consider that you know the role of divalent cations (Ca and Mg) in cells dissociation inhibition, we can briefly refer the problem to reducing trypsin efficacy due to repeated freeze-thaw or using very old dishes. Anyway you can change source of trypsin or playing with incubation time (20 mins or more).
Ali Moradi thank you for your suggestion I will try it, but don't you think the 20 minutes incubation isn't very long.
I don't know why when I recover the cells from Liquid Nitrogen I culture it and after 48 hours when I passage it, all the cells digest very well with in 5 minutes but when I culture it again and when I passage it on 2nd and 3rd passages its very difficult to digest even with 8 minutes incubation at 37C. I also tried different batch cells and trypsin.
If more mins doesnt harm to your cells let them treated longer time. With low concentration ( 0.125% or lower) of trypsin you can extend length of treatment time. Please check dishes every 2-3 mins under the inverted microscope untill the cells started to get round. Another factor affecting your work is residual proteins and divalent ions despite of initial cells rinsing. To get rid of them please wash away cells more (3 times) with PBS (without ca2+ and mg2+) prior to trypsinization.
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