Dear people,
We froze our THP-1 cells with 20% FBS and 20% DMSO. When we thaw our cells, about 95% of our cells die. Does anyone have a protocol which results in a higher viability?
Thanks in advance, I hope to hear from you.
Dear Dr. Behrouz Zandieh Doulabi
I prepare 2X concentrated stock freezing media, freshly prepared, and pre-cooled on ice for at least one hour before usage, which is finally diluted with the cell suspension.
Stock freezing media contains 60% complete cell culture medium + 20% FBS + 20% DMSO.
Final concentration after mixing cell suspension (in complete cell culture medium) with freezing medium at a 1:1 ratio will be 80% complete cell culture medium (containing cells) +10% FBS + 10% DMSO.
Protocol for thawing THP-1 cells.
1) You should follow a quick thaw process. Remove cryovial from liquid nitrogen storage and hold the cryovial on the surface of the water bath at 37 degree C, and occasionally flick it gently during thawing until only a small frozen piece of ice is still visible. Do not leave the cryovial unattended during the thawing process because thawing takes only a minute or two.
2) Decontaminate by spraying the vial with 70% Ethanol and transfer cryovial to a sterile hood.
All following steps should be performed under sterile conditions.
3) You may add warm complete media drop-by-drop into the cryovial containing the cell suspension, slowly over a 40-second period. The final volume should be around twice the volume of the cell suspension (for instance, add about 1 ml of complete media to a cryovial containing 1 ml of cell suspension). Be careful not to exceed the capacity of the cryovial.
4) Transfer the diluted cell suspension into a 15ml centrifuge tube containing 8ml of warm complete media. For cells preserved in a freezing medium containing DMSO, it is recommended to wash out or dilute the DMSO immediately post-thaw because DMSO harms the cells at higher concentration.
5) Centrifuge the cell suspension at low speed 250 x g for 7 minutes to prevent cell damage.
6) Decant the supernatant and tap the cell pellet.
7) Resuspend the cells in 20 ml pre-warmed complete media and transfer the cells into one T-75 cm^2 cell culture flask.
8) Incubate at standard growth conditions overnight.
9) THP-1 cells are sensitive to seeding density. Cells have to be sub cultured starting from 8 x10^5 cells/ml. Less concentrated cultures will stay under standard growth conditions for an additional 24 hrs.
Please note that freezing and thawing has an impact on THP-1 cell line more than other cell lines. Cells grow slowly and in aggregates during the first few weeks of culturing. Therefore, subculturing becomes necessary only once a week. Keep on subculturing until cells grow as single cell suspension without apparent aggregate formation (say at least for four subculturing cycles) before you use it for any experiment.
Good Luck!
Regards,
Malcolm Nobre
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