Protocol Online logo
Top : Forum Archives: : Cell Biology

THP-1 thawing and culturing protocol - (Sep/03/2008 )

can someone please give me an exact protocol for thawing from liquid nitrogen and cell culturing of THP1 cell?
This is my first time doing it and i'm not very sure what to do. I got this procedure below would it work? can you tell me what medium is needed and everything. from what i have read it's sounded really hard and need alot of care.

1. Recovery Medium: RPMI + 2mM Glutamine + 20% HI FBS
• To 0.8 vol. RPMI add 0.001 vol. uL 100X Glutamine and 0.2 vol. heat inactivated FBS
• Warm to 37oC

• Prepare 20 mL of RM.
• Wipe outside of vial with 70% ethanol.
• Place vial at 37oC until almost thawed.
• Transfer contents to a sterile 15 mL centrifuge tube.
• Slowly add 4 mL RM to the tube.
• Transfer 100 uL of suspension to a 1.5 mL microcentrifuge tube for counting
• Centrifuge the cell suspension at 100xg for 4 min.
• Decant medium and resuspend cells in 10 mL of RM (~ 5 x 105 cells/mL).
• Transfer to a T25 culture flask.
• Incubate flask in vertical position at 37oC:5% CO2 for 7 days.
• Check every couple of days (morphology and cell count).
• Lay in horizontal position for up to a further 7 days.
• Check every couple of days (morphology and cell count).



I'm using an easier protocol than this one for THP1 cells.

- prewarm RPMI, FCS and Glut at 37ºC.
- collect cells from liquid N2 with dry ice.
- put them in a water bath at 37ºC (let them thaw just a little)
- quickly centrifuge the vial at 300g, 5min, 4ºC.
- in a sterile hood, aspirate all the supernatant and resuspend them in 1ml of RPMI (containing Glutamine or adding it)+10%FBS+Pen/strep
- add cells to the plate (select the size of the plate depending on how many cells were frozen) containing the same medium.

And that's it! It works! They are not special cells at all.
I grow them in cell culture plates, since they are adherent it's easier to trypsinize them here than in a flascon. From the beginning, they are always in an horitzonal position.
The freezing vial can be directly centrifugated (it's quicker) to eliminate DMSO that kills cells. Put them in a falcon with medium previous to centrifugation is only to dilute the DMSO. I think it's better to centrifuge the vial directly.
In case you don't know the number of cells frozen, you can count them when you resuspend the cells in 1ml of complete medium.

I hope it helps!