Dear All, I am working on development of hybridoma technology. I was used balb/C mice for immunization and SpO cell lines as myeloma cells. After 3rd booster spleen was removed aseptically. fusion of mice cells with spo was carried out via PEG. The clone was screened after 14 days (HAT medium). The clon was pick from the well and performed serial dilution for single clone isolation. Now the clones are grow exponetially on HAT medium, But I am not getting antibodies in the medium.
Can any one help me...
Hi Anil,
New hybridoma clones are genetically unstable and can sometimes excise chromosomes. So your progeny may have lost the ability to make Ab.
Did you freeze prt of the oroiginal hybridoma before cloning? If so, you can go back and test it.
Good luck
Dear Anil,
I thing after HAT screening you should grow your hybrid in HT medium and transfer all properly grown positive hybrids to 10% medium in 12 well and then T-25 cm flask and check the secretion again and finally from T-25 flask go for single cell cloning.
Good Luck
Thank you Mr Brijesh for your nice suggestions. I have already tried to grow clone in 2% FBS containing HT medium. Clones are growing very well but i could not find out the antibodies. So is it possible, increasing conc of FBS will stimulate the antibodies secretion.
Thank You
I agree with Michael, and you should try to clone immediately after screening. Did you try to clone starting from 10 cells/well with step 3 dilution?
- Badges
- Science topic
- Similar topics
- Medicine
- Disease
- Infectious Diseases
- Immunization