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Freezing and thawing of cells - (Sep/09/2004 )

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QUOTE (mito1 @ Jan 17 2007, 05:51 PM)
QUOTE (penny @ Dec 6 2006, 11:18 AM)
Hi Scifi,
I am sorry I did not check out this thread for a while.
But I think, that it is best to place plain media without FBS in incubator for 30 minutes at the max.
That balances pH (due to the CO2)...take out flask, add right amount of FBs( generally 10%) and then thaw the cells and add suspend them in this media.
I have personally tried with and with out FBS and this works the best.
Penny




QUOTE (scifi @ Nov 29 2006, 10:27 PM)
QUOTE (penny @ Feb 7 2006, 03:31 PM)

Hi Everyone!
I have yet another question.. what is cause of heat inactivation of FBS..... before thawing or subculturing..I place the culture medium(incl. FBS) in the incubator(5% c02 at 37 degrees) for an hour. Could it lead to FBS inactivation?
Penny


Hi Penny,

Did you get a reply for this question - Placing all the ingredients in the incubator for ~1hour or so - will this lead to FBS inactivation? I too do this quite often. Is this question answered specifically? In this thread I did not find an answer to this.

Thanks.




Hi,
If I am reading this right, and I may not be (as the coffee has worn off closedeyes.gif ), it seems that 37 degrees for 1 hour should NOT inactivate the serum: after all, the cells grow in this media, sometimes for a few days. To actually heat-inactivate serum, you need temperatures around 56 - 60 degrees.
mito 1



Classical heat inactivation of FCS/FBS is 30 minutes at 56oC. This was done years ago to inactivate compliment and to reduce the possibility of mycoplasma contamination. With filtration of serum these days (0.04uM) this is no longer necessary. It is even thought that prewarming media at 37oC is enough to inactivate heat-labile compliment components.

-Rhombus-

QUOTE (mito1 @ Jan 17 2007, 04:51 PM)
QUOTE (penny @ Dec 6 2006, 11:18 AM)
Hi Scifi,
I am sorry I did not check out this thread for a while.
But I think, that it is best to place plain media without FBS in incubator for 30 minutes at the max.
That balances pH (due to the CO2)...take out flask, add right amount of FBs( generally 10%) and then thaw the cells and add suspend them in this media.
I have personally tried with and with out FBS and this works the best.
Penny




QUOTE (scifi @ Nov 29 2006, 10:27 PM)
QUOTE (penny @ Feb 7 2006, 03:31 PM)

Hi Everyone!
I have yet another question.. what is cause of heat inactivation of FBS..... before thawing or subculturing..I place the culture medium(incl. FBS) in the incubator(5% c02 at 37 degrees) for an hour. Could it lead to FBS inactivation?
Penny


Hi Penny,

Did you get a reply for this question - Placing all the ingredients in the incubator for ~1hour or so - will this lead to FBS inactivation? I too do this quite often. Is this question answered specifically? In this thread I did not find an answer to this.

Thanks.




Hi,
If I am reading this right, and I may not be (as the coffee has worn off closedeyes.gif ), it seems that 37 degrees for 1 hour should NOT inactivate the serum: after all, the cells grow in this media, sometimes for a few days. To actually heat-inactivate serum, you need temperatures around 56 - 60 degrees.
mito 1



This is true to my knowledge also. In order to heat inactivate, we need to place the FBS in water bath set at 56 C for 40-45 minutes. This will inactivate some of the components in FBS. But we also did a comparative study by growing some cells in heat inactivated and non-heat inactivated FBS containing media. Result = no noticeable difference. Anyway, this depends on the cell line in question.

-scifi-

QUOTE (humab @ Mar 15 2005, 12:57 AM)
while i think it might be difficult to agree on a procedure that will work best for most, please make sure NOT to

QUOTE
take the viall out still with some liquid nitrogen, walk to the water bath.
!!!

most cryovials can - under certain circumstances - let some of the liquid nitrogen leak into them. any cryovial coming from liquid nitrogen storage is therefore to be considered dangerous and explosive for the first few minutes. take it out of the tank using usual safety precautions, place it into a special container, and let it sit for a few minutes. THEN take it to the water bath.

This is not hearsay, it has happened to me more then once!

take care.m

didn't know that
thanks for the note, I will keep it in mind when I handle the "dangerous" vials! unsure.gif

-Lynnpanda-

hi
I need help, I got trouble with my cell lines, they dont grow or attached to the flasks. Ill been reading the chat about thawing and preseving cells, I do the same told by the protocol online web, but we try to diferente thawing methods.
One we thaw with 37┬░ bath water for 2 min, add new MEM 20%fetal bovine
serum to inactivate DMSO, than centrifugate at 1500rpm for 10 minutes and resuspend with nen MEM 20% and put them on the flask, Initially we observed round and brilliant cell thay float free of the surface. The next day we got a little attachmente and after 2-3 days somethings is wrong and they die... }
The second method differnce couse we thaw cells in cold water until the ice thaw and imidiatlly centrifugate them 1500 rpm for 10 minutes, then add MEM 20% FS, and put them in the flask... this seems to work better, cells attached faster but indeed after 3-4 days they die. And I dont know why... need help please

Mcrb. Pamela Apolo

JB Pharma
Biol├│gicos
Quito-Ecuador
pamelula@hotmail.com

-PAME-

I wouldn't thaw cells in cold water. Always thaw cells in 37 degree C waterbath. You can't really inactivate DMSO but you can dilute it to a concentration (< 1%) which is not toxic to cells.

You can try serum from other source. You can add cells to a medium and seed them in a flask without centrifuging them.

-exploresci-

QUOTE (exploresci @ Jun 8 2007, 07:48 AM)
I wouldn't thaw cells in cold water. Always thaw cells in 37 degree C waterbath. You can't really inactivate DMSO but you can dilute it to a concentration (< 1%) which is not toxic to cells.

You can try serum from other source. You can add cells to a medium and seed them in a flask without centrifuging them.


Hello everyone,
I'm working on Drosophila S2 cells and have established stable cell lines.
I prepared the frozen stocks by spinning down my cells at 200 g for 10 mins and resuspended the cell pellet in a media, which contain 45% conditioned media, 45% fresh S2 media and 10% DMSO. Then aliquotted in to cryovials, kept at 4 C for 30 mins and transferred to -80 C overnight and then transferred to liquid nitrogen.
To thaw: I thawed the cells at 37 C and then transferred the cells to 5 ml of fresh S2 media with blasticidin at a concentration of 10 ug/ml. Even after a week, my cells didnot recover and the cell count is just 0.5 million cells/ml. I used the same procedure for the S2 cells and it worked. But its not working for my cell lines.
Is there anything wrong in my procedure??? If so, could anyone please correct me and gimme some suggestions

Thanks

-Nadella-

Hey,

I know they're are always ten ways of doing somethings, but our lab uses DMSO. We freeze down our cells in an alcohol containing cryopreserving container in our -80 overnight. It's just 70% ethanol bathing a tube rack. Works great, never an issue with culture media +20% FCS +10% DMSO.

-PhD Wannabe-

hello!!!

try to reduce centrifugation speed.

-rox-

i have a Q....

wht is the best method to freez 300 Million cell from a buffy coat ....like shall i devide it in 3 tubes and freez seperatly ....or in one go?

-Muna-

and also ...the pellet size get reduced from wash to wash ....some time it is 1/2.... blush.gif
....the speed i use, is realy high 300g..can be the reason.... excl.gif

-Muna-

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