Reviving THP1 cells - (Jul/06/2008 )
Hiii everyone..
I have been working on THP1 cells for the past one year. Right now I am trying to revive THP1 cells from Liquid nitrogen stock. This is exactly what I do..
1. Thaw the frozen stock in warm water by gently swirling
2. In the hood, transfer the contents after pipeting once gently to a 15ml centrifuge tube
3. Add 9 ml of RPMI mediium with 10% FCS, drop wise.
4. Spin at 1300 rpm for 10 mins
5. Decant the supernatant and resuspend pellet in fresh medium
6. Check for viability by trypan blue exclusion test.
7. The cells are then incubated in 5% CO2 incubator in a 6 well plate.
I have followed the above protocol and tried reviving the cells 4 times. 3 out of 4 times I found the cells to be all dead when tested with trypan blue. Once I got 10% viable cells, and when I tried to grow them, after 5-6 passages, the cells stoped growing.
Can anyone tell me the following
1. Where could be the problem in reviving cells since I get all blue cells when i do the trypan blue test after reviving
2. Once the thawing is done, how to proceed with the subsequent passages to get a healthy, growing cells.
Thanks
JEM
I have been working on THP1 cells for the past one year. Right now I am trying to revive THP1 cells from Liquid nitrogen stock. This is exactly what I do..
1. Thaw the frozen stock in warm water by gently swirling
2. In the hood, transfer the contents after pipeting once gently to a 15ml centrifuge tube
3. Add 9 ml of RPMI mediium with 10% FCS, drop wise.
4. Spin at 1300 rpm for 10 mins
5. Decant the supernatant and resuspend pellet in fresh medium
6. Check for viability by trypan blue exclusion test.
7. The cells are then incubated in 5% CO2 incubator in a 6 well plate.
I have followed the above protocol and tried reviving the cells 4 times. 3 out of 4 times I found the cells to be all dead when tested with trypan blue. Once I got 10% viable cells, and when I tried to grow them, after 5-6 passages, the cells stoped growing.
Can anyone tell me the following
1. Where could be the problem in reviving cells since I get all blue cells when i do the trypan blue test after reviving
2. Once the thawing is done, how to proceed with the subsequent passages to get a healthy, growing cells.
Thanks
JEM
Dear JEM,
Just revived one of our THP-1 vials last week, within 3 days enough cells to split (95% viability).
Can I give some suggestions:
a). When the cells were originally frozen, did anyone try to revive them later to check for viability?
. I warm the vial in my hand, only adding fresh media when the ice in the vial is just about to thaw....THEN CENTRIFUGE AT 700RPM for 5 MINUTES........ very important to reduce down your speed....1300RPM is far to fast and far to long just to pellet your cells. ALSO USE MORE MEDIA....about 30-40mls.c). USE THE BEST QUALITY SERUM...what do you use?
d). Put the cells into a T25cm flask.... this will give them the best chance... i.e. it is better to have too high a cell density than too few.
e). Do not use plates to grow cells.....take advantage of FLASKS THAT HAVE 0.2uM FILTER TOPS....this will drastically reduce the risk from contamination.
Hope this is useful.
Kindest regards.
Rhombus
It sounds like they were not frozen down properly, so their viability is very poor. The most important thing when you thaw cells is _speed_. You have to do it quickly. Of course it depends on the cell type, but since the provided (slow) protocol does not help you much, try to:
- thaw in water bath
- when you see the piece of ice detaching from the tube, you can wipe the outside of the tube with ethanol, and simply pour the liquid and ice into the volume of medium you need (no dropwise addition, no centrifuging and stuff)
- Make sure that the DMSO is diluted to lower than about 0.5% in the end volume. It will not cause any problem at that concentration
- Change medium after the cells attached (to get rid of dmso in case it does cause a problem
)
Works for me.
- thaw in water bath
- when you see the piece of ice detaching from the tube, you can wipe the outside of the tube with ethanol, and simply pour the liquid and ice into the volume of medium you need (no dropwise addition, no centrifuging and stuff)
- Make sure that the DMSO is diluted to lower than about 0.5% in the end volume. It will not cause any problem at that concentration
- Change medium after the cells attached (to get rid of dmso in case it does cause a problem )
Works for me.
Dear Kupac,
THP-1 should always be frozen down in Glycerol and not DMSO, unless you want to use diffferentiated THP-1 cells?
Rhombus
I never worked with these cells, but the principle is the same. Be quick.
Dear Rhombus,
Here in our lab the Thp1 cells are always frozen in DMSO. Few experiments we do without differentiating it. For one expt we do differntiate those cells to macrophage-like cells. Is there any specific reason as to why we need to freeze them in glycerol
JEM
- thaw in water bath
- when you see the piece of ice detaching from the tube, you can wipe the outside of the tube with ethanol, and simply pour the liquid and ice into the volume of medium you need (no dropwise addition, no centrifuging and stuff)
- Make sure that the DMSO is diluted to lower than about 0.5% in the end volume. It will not cause any problem at that concentration
- Change medium after the cells attached (to get rid of dmso in case it does cause a problem )
Works for me.
Dear Kupac,
THP-1 should always be frozen down in Glycerol and not DMSO, unless you want to use diffferentiated THP-1 cells?
Rhombus
Hey JEM...a little late for this one but i work with THP-1 cell line.
Well cryopreservation procedure is almost the same for all cell lines...we use DMSO-FCS for long term storage, but by hit and trial i personally found the following protocol simple and effective for reviving THP-1.
1) remove cells from liquid N2 and thaw them by swirling in warm water (usual)
2) then without any centrifugation culture entire contents of the cryovial (storage medium+cells) into RPMI medium and forget about it. keep in incubator for a couple of hours.
The idea is to just acclimatize the cells to the new environment, since we know how sensitive THP-1 cell line is to any kind of physiological stress.
3) after say 2 hrs centrifuge and pellet the cells out, i pellet cells at 2000rpm for 5 minutes or 1000rpm for 10 minutes and then culture them as usual in fresh media.
WORKS PERFECTLY WELL FOR ME!!!