THP-1 cell line - (May/10/2011 )
I had taken one vial of THP-1 cells from liquid nitrogen for culturing but it is not growing. I have already wasted 4 vials.
Kindly let me know what should I do so that the cell start to grow. I need them for my experiment.
Do you know the density of the frozen stock? Try growing them in a T25 first and moving them to a T75 after they have bulked up a bit.
What protocol for defrosting are you currently using?
Hi there. I just brought up a new cryovial of THP-1 cells a week ago. Quick question first though: How were they frozen down? Resuspending your cells in 90% serum and 10% DMSO before freezing seems to be the best way to go in terms of protection and recovery. DMSO can induce differentiation BUT it protects the membrane much better than glycerol and because the exposure time is so low differentiation of the THP-1 cells is not a problem.
When I take the vial out I let it 1/2 thaw in a water bath and then transfer it to 4 mL of recovery media in Recovery Media(RPMI media, serum, antibiotics and extra L-glutamine) and centrifuge it at 100xg for 5 minutes. Resuspend it in 8mL of the recovery media and transfer it to a T-25. I let this sit in the incubator (37C, 5%CO2) for a week checking cell count and morphology every other day. Passage your cells at least once after this before using them.
Hope this helps! If the cells were poorly preserved this could be a major factor in their inability to recover and grow.