M-NFS60 cell line revival - Revival protocol (Aug/21/2006 )
Hello,
I am procuring NFS60 cell line from ATCC , but failed to revive it.
The method I follow is : Upon receiving it I store it at liquid nitrogen. Next day from liquid nitrogen I remove the vial & thaw it at 37oC water bath. After decontaminating it with ethanol I transfer the contents to T75 flask ( tissue grade nontrated )contaning the growth medium & incubate it at 37OC +%% co2.
No growth was observed & viability also decrease ( trypan blue staining).
Please help.
Was the culture still frozen when you received it? If it had thawed that would explain cell death.
Assuming it was received in good order, did you wash out the DMSO immediately after thawing your cells?
Assuming it was received in good order, did you wash out the DMSO immediately after thawing your cells?
The order was received in good condition (in frozen form).
No, I did not remove cryomedia immediately after thawing, I remove it after 24 hrs. But the viability of cells decreases as time proceed.
Please help.
Nim10,
There is a whole thread on this forum about thawing cells, but I cannot believe that you were taught NOT to remove the Cryopreservative from the cells. The person who told you that should be shot, you have just wasted £200-£400 on the cell line you bought from the ATCC and your time as well.
Basically cells are frozen in 10% DMSO and at that concentration DMSO is cytotoxic i.e all your cells will die.
The level of DMSO to achieve is below 0.1% i.e a 1:100 dilution of the concentration in the vial. Preferably it should be 0%.
This can be achieved in 1 of 2 ways:-
i) Thaw vial, add media, spin at 150g, resuspend in fresh media and plate onto TC flask/dish......this is the PREFERED METHOD everyone uses.
ii) Thaw cells, dilute with media, add to flask, allow cells to adhere, wash off media containing DMSO and add fresh media........ If you have a 1ml cell suspension from the ATCC, you must diltue it 1:100 to achieve 0.1%...i.e. waste 100mls of media....NOT THE PREFERED METHOD.
Some people will argue that centrifugation damages cells...it does. However DMSO is more damaging.