There are many different medium combinations for hybridoma expansion. I have conducted fusion of Sp2/0 Ag 14 cell line with B cells from balb/c mice. I am using DMEM-20/ 10 mM HEPES/1 mM sodium pyruvate/1xHAT. I would like to get advice about the medium suitable for expansion of cell line. Will it be suitable to proceed with the thymus cell extract from balb/c mice. Kindly help me.
Sounds like you have difficulties in limiting dilution of hybridoma cells. Many many years ago, we had used feeder cells such as fetal thymocytes or peritoneal cavity fibroblasts during limiting dilution. However, these days, we do not use feeder cells. I think your problem is not due to the medium of your choice. My suggestion is 1) check whether your Sp2/0 cells are infected with mycoplasma. You can purchase medium containing drugs that eliminate mycoplasma. 2) before fusion, you had better to do limiting dilution of Sp2 parental cells to get rapidly growing clones in your medium.
Dear Professor, Thank you very much for the valuable instructions. Further, will you tell me, during the expansion step, is it essential to use a supplements such as, hybridoma cloning factor (HCF), in order to enhance the viability and rapid expansion of antibody producing cell.
Thank you.
Dear Dr. Uede, what do you mean by limiting dilution of parental cells? Is it performing a let's say 1:5 dilution 24 h before fusion?
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