Reviving a hybridoma from LN2 - (May/20/2010 )

Hi all,

We're about to revive some frozen hybridoma cells and the procedure recommends co-culturing with normal mouse spleen cells until the hybridoma cells can proliferate on their own. I'm having trouble finding a source of normal mouse spleen cells.

1. Is it necessary to co-culture?

2. Sources of normal mouse spleen cells?

3. Alternatively I think we can just use conditioned medium but the source I found (Sigma) has it back ordered until July. Any other conditioned medium sources?

Thanks!

-P

We just added some IL-6 to some of our lines to kick-start them. I don't recall co-culturing.

Sometimes we co-culture with normal mouse spleen cells, and sometimes we don't. We do, however, make them ourselves. Just sacrifice a "normal" (non-stimulated for Ab production) mouse, preferably the same strain, and dissect out the spleen. Collect/extract the spleen cells the same way you would for a hybridoma fusion (including some washes). Resuspend in medium and plate out. Typically, we do this the day before we need them.