Attention FGARBREC (sorry, I dumped your original post and can't
remember your name)! Your reply address doesn't work.
My original message, which has been reflected back by your mailer 3
times now, was:
> I have, over the past couple of years, gotten a number of murine
> hybridomas to grow well in various serum free media, but have been
> unsuccessful in cryopreserving serum-free adapted hybrid cell lines.
> I use the standard 10%DMSO/90%FCS as a cryopreservative,
This is really strange. When we were generating MAb hybridomas
(mouse-rat fusion; really tough), we could both adapt what few
hybrids we got to serum-free and were able to successfully freeze and
thaw whenever we felt like.
Our freeze-down method is slightly different. There are two
cryopreservatives:
Freezing Media Cell Media
10% DMSO 80% FCS
70% FCS 20% DMEM or RPMI 1640
20% DMEM or RPMI 1640
Everything is filtered through Nalgene 0.22 um nylon units. Freezing
Media and pipets for it are put into -20oC freezer 30 min before use.
Cells are harvested as usual and resuspended in Cell Media at approx.
2 to 3 million/mL. 0.5 mL are then loaded into each CryoNunc vial (1
million/tube). 0.5 mL Freezing Media is then dribbled down the sides
of each vial (if you can make a layered drink or load Ficoll
gradients from the top, you can do this).
CryoNuncs are kept at -70oC overnight and then transferred to liquid
nitrogen. We used to use a vapor system but switched to a liquid
suspension system because it was cheaper.
If you have any problems or comments, please call or write; my phone
number is on the signature. Have fun.
- ivan
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Ivan Shaw "The Angel of Death"
Neuroimmunology Unit
Montreal Neurological Institute
Montreal, Canada H3A 2B4
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Internet e-mail: Ivan at MNI.LAN.McGill.CA
Telephone: (514) 398-3002
FAX (514) 398-7371
