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Message | User | Date(yyyy-mm-dd) |
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HL-60 revival problem | neelimak | 2008-09-27 | Click here to register. |
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Hi,
Iam not able to revive HL-60 cell line after freezing.All cells are dying after revival. I tried using DMSO and glycerol with 90% serum. Please give me a suggestion | | |
| lubrol | 2008-09-29 | Click here to register. |
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 | Neelimak;
First a few questions and some tips that might help. It is hard to be specific as I don't know the details of your procedure.
1. How are you thawing the cells? They should be thawed fairly rapidly in warm water and then immediately diluted into pre-warmed medium plus serum and spundown gently.
2. How are you freezing the cells? They should be frozen at cooling rate close to 1 degree C per minute.
3. What concentration of cells are you freezing? Typically the best results are obtained with a concentration around 2-3 million cells per mL.
4. Were the cells frozen in liquid-phase Nitrogen, vapor-phase Nitrogen or in an ultra-low mechanical freezer? They really need to be stored below -135 degrees C for optimal recovery and below -150 is better.
5. Were the cells checked for bacterial and mycoplasmal contamination prior to freezing? Cell lines that can tolerate mycolplasma infections prior to freezing, may be too stressed from the freeze/thaw proceeedure to survive the infection.
You can find some good tip at:
http://tinyurl.com/5x7mbs
http://tinyurl.com/3p9bwp
and http://www.nuncbrand.com/us/frame.aspx?ID=608
They all have manuals that describe basic freezine techniques.
Hope this helps.
Lubrol |
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| neelimak | 2008-09-30 | Click here to register. |
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| Hi,
First of all Thanks for your reply.Iam following all precautions as you mentioned. Here iam expalaining the procedure what we are following in our laboratory.
We are facing problem with HL60 cell line in freez thawing. Cells would
be fine untill they are in running culture, but once after freez thaw
its not getting revive completely, not even 20% recovery. Even with THP1
also we had problem but atleast few of the batches could able to
recover. I'll just breifly explain you the freezing and thawing
procedure that we follow here..
Steps in Freezing:
1. Pellet the cells at 1000rpm for 10 mins.
2. Take the cell count and prepare the freezing media (90%FBS+10%DMSO) and
alloquot in vials with 3million no. of cells, place in cryo cooler
containing isopropanol.
3. Place the cyro cooler in -70C for overnight and transfer them in Vapour
phase of
liquid nitrogen.
Steps in Thawing:
1.Take the vial from Liquid nitrogen tank and gently thaw at 37C
2. Remove the cell suspension, and put in 50ml falcon tube conatining
around 9ml of
growth medium, pellet for 10 mins @1000rpm.
3. Resuspend the cells with growth medium(RPMI+20%FBS) and put appropriate
no. of
cells in pre-warmed flask, incubate at 37C+5%CO2.
If any modifications or extra precautions that we need to take please let
us know.
Looking forward for your valuable suggestions.
once again thanks for your reply,
Regards,
neelima
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| lubrol | 2008-10-08 | Click here to register. |
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| Hello again;
Thanks for the information on your procedure, it helps.
Looking at what you do there are only a couple of things that stand out as possible sources of concern to me. First, 10 minutes at 1,000 RCF is a bit long; I wouldn't normally expect that you would need to centrifuge the cells for more than 1 or 2 minutes; this could be an real issue especially after thawing. A second issue maybe the use of 90% FBS plus 10% DMSO. These cells can be induced to differentiate by the presence of DMSO so you might be better off with 5%. Also, using complete medium containing 20% FBS instead of pure serum might help.
Through all of this, I'm assuming that the cells have been frozen and recovered from vapor-phase storage before and that there is no mycoplasma contamination of the line.
One other question: do you know how many passages the cell line has seen both with respect to the cultures you are trying to freeze and any other vials that might be frozen away with fewer passages?
I notice that you are using RPMI (1640?) for propagation. As these are not a truly adherent cell line, I would have expected that Iscove's DMEM or another slightly richer medium like Alpha MEM designed for less adherent cells. But, if you have been successful with RPMI that's a fine medium once the cells adapt to it.
Do you known what the viability of the cells (% viable) is prior to freezing and immediately after thawing via Trypan Blue, Alamar Blue, Neutral Red or other technique? It seems strange that you are having trouble as the protocol looks pretty good and these cells should freeze and recover fine.
I'm a bit perplexed but hope some of this will help.
Lubrol |
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| neelimak | 2008-10-13 | Click here to register. |
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| Hi,
Thanks for your help lubrol. Last week I tried with 95% serum and 5% DMSO. Luckily it worked out. But iam not sure whether i will be successful every time. Iam trying with different batches.
Iam working with lower passages i.e below 40 passage.
Yes iam taking vaibility count before and after freezing too with trypan blue.
Any way thanks once again for your help. I will be keep trying till i succeed. I will let you know my results in my next mail.
Thanks lubrol.
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