| |||||||||
| Login :: Register :: Search forums :: Top Users :: Reagent |
|
Position: Discussion forums > Bioscience biotechniques discussion forums > Immunology forum and cytometry forum
|
hybridoma madness...
Posted by: aquavet (IP Hidden, New member, 1)
Date: April 17, 2006 02:01PM
We are trying to produce MAbs agaist one virus. This is the 4th fusion and we don't even get to the point of cloning our cells as they die all before...
The Balb/c are were injected with purified virus in MPL-TD adjuvant (3 times at 3 weeks interval) they received a booster (no adjuvant) IP 72 hours prior to the fusion. The fusion partner are SP2 cells that are treated with 8-aza for 1 week, then put in complete media (at least 1 week prior to the fusion). The SP2 are splitted 24 hours prior to the fusion. The mouse are killed with halothane and the fusion is done using PEG according to protocole that seem to be fairly standard. We use 1X10E7 Sp2 cells per spleen. The cells are counted just prior the fusion by tryptan blue exclusion. Just after the fusion the cells are put into conditionned media and then we add 2XHAT (4ml in 96ml complete media) 24 hours following the fusion and the "hybridomas" are fed again at day 6. We do have a lot of nice happy looking cells the day following the fusion but then it goes down hill. The cells do not form cluster and slowly die off. (Now it has been 1 week post-fusion but the pattern was fairly similar for all the previous fusion). There is no bacteria contamination in the wells (visual + PCR using bacterial primer on the supernatant). The SP2 were assayed for mycoplasma and were found negative. We have switched to plasticware, gotten new reagent (HAT, HT, 8-aza) and the hood was fumigated. We sort off run out of ideas for the troubleshooting. Anyone has any insight? Have you ever seen something like this? thanks!
Re: hybridoma madness...
Posted by: Paul B (IP Hidden, New member, 1)
Date: June 8, 2006 02:49AM
You could try a media supplement called "Briclone" available from NICB, Dublin City University, Glasnevin, Dublin 9.
Lots of companies use this supplement to support early hybridoma clones until they have been selected & screened. Drop a mail or call AnneMarie Larkin annemarie.larkin@dcu.ie 0035317005401, She is the head of Immunology for the NICB and specialises in hybridoma generation, selection etc. Alternatively try Donnacha O'Driscoll donnacha.odriscoll@dcu.ie 0035317005283, he was involved in the development of Briclone and might provide you with information to decide whether it is appropriate for your application. Their advice will cost nothing and I'm sure that AnneMarie in particular will put you on the right lines, just mention that you were in contact with me and I'm sure that they will help you out. Good luck Paul
|
We are moving ... Please post to our new community forums
|
|
||||||||