I am working on monoclonal antibody production. I use the SP2/0 cell line. 5 days after fusion I change the medium with fresh fusion medium in HAT, after 6 days I see one or more clusters of cells in each well. Also on the seventh day, I change the medium with fresh fusion medium in HAT. I see my well on the ninth day. Unfortunately, all of the clusters of cells not divided and most of them died. what is the problem in my fusion? I sure about PEG and FBS. Is it possible that these clusters of cells have not been hybridoma clones and are sp2/0 or lymphocyte?
In this situation should I change the medium or not?
Hi Nasim,
I would recommend adding the HAT in your fusion media and leave the hybridomas undisturbed for 10 days in the incubator after the fusion. If you can, use an incubator where only the hybridomas are being grown; if other cell lines are cultured then the door will be opened/closed quite often causing a lot of disturbance to your freshly fused hybridoma (they are very fragile cells).
Also do you use feeder cells or some kind of hybridoma growing supplement?
We use BriClone or GroClone ( https://nicb.ie/briclone/) at 5% for all our hybridoma cultures during and post fusion; these supplements are easier to use than the feeder cells and provide a fantastic growth support.
I hope this helps!
Thank you for your recommendations
But, I explained incomprehensibly.
I added the HAT medium the day after fusion. Also, I use feeder cells. I did not take the plates out of the incubator for 5 days. I changed the medium on the sixth day after fusion. then I changed the medium every other day. Unfortunately, I can't use a specific incubator.
My question is why the colonies didn't stable and proliferate.
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