hybridomas

alustig at home.com alustig at home.com
Mon Nov 23 22:01:06 EST 1998


Freezing at -80 is not the optimum.  They should have been kept in
liquid nitrogen.  Either way, hybridomas tend to be pretty resilient and
they should still be able to revive after two years.  Sometimes they may
take a couple of weeks to "wake up". Hybridomas tend to be fast growers,
although sometimes if they're making a lot of antibody, they may be a
little slower.  On the average you can expect a doubling time of a few
days.  The cells should be nicely rounded. When you first thaw them, do
so into whatever volume they were in when frozen.  Let them rest
overnight. If after a day or two they look rough-edged, have a lot of
debris, or have a lot of dead cells by trypan blue exclusion, then just
leave them alone for a few days to a week.  After that time, do a gentle
split such as 1:2 every few days.  After a couple of changes, if they're
going to survive, you'l see some rounded healthy cells begin to emerge.
Good luck.

Gul Afshan wrote:
> 
> I am trying to wake up hybridoma cells produced by the fusion of mouse
> splenocytes to myeloma cells (Sp2/O-Ag14). The hybridoma cells were selected
> for HGPRT+ and cryopreserved at -80C almost 2 years ago. Since this is my
> first time working with hybridoma cells, I would appreciate any guidelines
> for how I can tell if the cells have been succesfully revived (morphology,
> growth rate, etc.).  They appear to be growing very slow, for they do not
> reach confluency after a month of growth in 5% CO2, 37C, in Opti-MEM (+5%
> FBS) in a T-75 flask.
> 
> Yuki Ohigashi
>



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