I am culturing a murine b cell hybridoma in RPMI with 10% FBS, 1% pen/strep and 1% L-Glutamine. However when I count them on a haemocytometer with trypan blue the vast majority appear dead. I have tried doing a low velocity spin to remove the dead cells however this doesn't help. Does anyone have an ideas why my viability is so low or how to increase it?
Christopher Davis, we are either co-cultivating our hybridomas with feeder cells (in our case: thymocytes from neonatal mice) or we use their supernatant to stimulate growth of hybridomas. Feeder cells should express high amounts of IL-6.
hi
There are several possibilities :
1- hybridoma clone has not reached the stability stage yet.
2- infected hybridoma with microbe ( especially mycoplasma )
you should check No 1 & 2 , if your clone was stable and mycoplasma free >>
passage the cells at high density in RPMI 20% medium .
good luck
Dear Christopher Davis
I would suspect that your hybridoma may be infected. Please see the QC process below, which common infections with mouse pathogens affect your hybridoma cultures.
https://www.sdix.com/products-services/contract-manufacturing/cell-line/
In addition, please check their elaborate FAQ section, which offers great advice for hybridoma storage and culture. As their labs are ISO certified, I suppose their QC is top notch.
https://www.sdix.com/products-services/contract-manufacturing/monoclonal-faq/
All the best & good luck with your experiment,
Michael
- Badges
- Science topic