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Recovery of HeLa cells - after thawing recovery is 0% (Jan/25/2006 )

I am using 5% DMSO in 100% FCS as freezing solution. After pelleting the cells, i suspend the cells in this freezing solution and then store them at -20,

when i tried to revive cells after 15 days, by thawing the cells at 37 for 30 to 60 seconds, i had added the cells (1ml) to pre-warm medium (5ml) in 25 cm dish.

after reading couple of notes on this forum itself, i realised that i shd have added cells dropwise to the medium, however my cells just die within 24 hrs.

my other question is, if we see black (very) tiny spots or black spikes on top of the adhered cells ((floating))....in the dish ...what does this signify ???

is it bacterial contamination ....but since i am cseing the cells beneath these strange dots and spikes, i am not sure ....please advise

Thanks,
jhil

-jhilmil-

Media should be crystal clear, even more if its hela. tiny spots moving might be contaminants, see their movement with highest magnification.
after resuspending the cells in 5 ml media i centrifugue again, discard supernatant and resuspend in 10 ml of fresh media to get redeemed of DMSO (very toxic)


QUOTE (jhilmil @ Jan 25 2006, 04:56 PM)
I am using 5% DMSO in 100% FCS as freezing solution. After pelleting the cells, i suspend the cells in this freezing solution and then store them at -20,

when i tried to revive cells after 15 days, by thawing the cells at 37 for 30 to 60 seconds, i had added the cells (1ml) to pre-warm medium (5ml) in 25 cm dish.

after reading couple of notes on this forum itself, i realised that i shd have added cells dropwise to the medium, however my cells just die within 24 hrs.

my other question is, if we see black (very) tiny spots or black spikes on top of the adhered cells ((floating))....in the dish ...what does this signify ???

is it bacterial contamination ....but since i am cseing the cells beneath these strange dots and spikes, i am not sure ....please advise

Thanks,
jhil

-tertu-

I agree with Tertu, resuspend your cells and centrifuge! Some colleagues had a very low recovery with Hela cells and I told them to centrifuge and their recovery was a lot better!

Apart from this: are you really storing your cells in -20? You should store in liquid nitrogen which is -196.
I'm not an expert, but at -196 there's hardly anything moving, while @ -20 some stuff still moves around destroying your cells (it's not Ice @ -20, but more of an amorfic glass situation maybe, not 100% sure.

-vairus-

hey,

I freeze my cells mammalian cells in DMEM 20% serum 10% DMSO but these percentages in my experience are not critical for most sell types, and using 100% serum is also OK.

One potential reason you are having a bit of trouble is that you are not getting the DMSO out of the culture after you are thawing your cells. As the previous post mentioned, you can add the thawed freeze down to 10Mls then spin them out and resuspend then plate. Or you can just add your thawed freeze down to 15 Mls of your grow medium and skip the spin out.

Another potential issue is how healthy your cells are when you freeze them down, I split a confluent plate 1 to 2 and freeze down the following day. Black spots/spikes could be cell debris.

hope it helps,

jeff



QUOTE (tertu @ Jan 25 2006, 12:21 PM)
Media should be crystal clear, even more if its hela. tiny spots moving might be contaminants, see their movement with highest magnification.
after resuspending the cells in 5 ml media i centrifugue again, discard supernatant and resuspend in 10 ml of fresh media to get redeemed of DMSO (very toxic)


QUOTE (jhilmil @ Jan 25 2006, 04:56 PM)

I am using 5% DMSO in 100% FCS as freezing solution. After pelleting the cells, i suspend the cells in this freezing solution and then store them at -20,

when i tried to revive cells after 15 days, by thawing the cells at 37 for 30 to 60 seconds, i had added the cells (1ml) to pre-warm medium (5ml) in 25 cm dish.

after reading couple of notes on this forum itself, i realised that i shd have added cells dropwise to the medium, however my cells just die within 24 hrs.

my other question is, if we see black (very) tiny spots or black spikes on top of the adhered cells ((floating))....in the dish ...what does this signify ???

is it bacterial contamination ....but since i am cseing the cells beneath these strange dots and spikes, i am not sure ....please advise

Thanks,
jhil

-jsavas-

Hi,

here is what i do with all my cell lines:

-after one wash in culture medium, pellet are resuspended in DMSO:FBS 10:90 (at 5 to 10e6 cells/ml), for some days at -80C and then for long time in liquid nitrogen

-i thaw the cells by adding medium directly on frozen cells (on "ice cube"), pipete the thawed cells and dilute them in medium, and repete till all the ice has melted, in order to avoid too long contact with DMSO; one wash; 24h culture and then another wash

-if growing difficult, try anti-mycoplasma

= always ok wink.gif

S├ębastien_

-tryptofan-