I am standardising the methodology for hybridoma production. I did fusion with PEG and cultured in HAT supplemented DMEM+10%FBS. after five days I found several cluster of cells in HAT supplemented DMEM+10%FBS. after 10 days of fusion I transferred these cells into HT supplemented DMEM+10%FBS. Unfortunately, the cells didn't grow even after 10 days. it looks like same that I inoculated. what is the problem in my fusion? how should I improve the growth?
I have seen this before using Sp2 myelomas. I would reccomend you up you richness of your media by either adding more FBS and or Hybridoma cloning factor. Once they are off you can wean off cloning factor.
I usually change from HAT to HT media not too late as 5th day, but I always use RPMI 1640 and X63Ag8. Please read this paper for more information. Hope you'll be successful next time.
https://www.researchgate.net/publication/356570735_Murine_monoclonal_antibodies_against_RBD_of_the_SARS-CoV-2_spike_protein_as_useful_analytical_tools_for_subunit_vaccine_development_and_clinical_trials
I use RPMI 20% FBS + supplements for hybridoma culture.
If your hybridomas grow poorly, I highly recommend you to change HAT to HT medium following bellow steps:
Day 0: Fusion -> Day 1: Adding HAT medium -> Day 7: Changing HT medium (1st time) -> Day 9: Changing HT medium (2nd time) -> Day 10: Screening
Please note that checking regularly could affect hybridomas growth. Just let them be stable in incubators.
Andy Giovel Domínguez Rodríguez
thanks for your suggestion. did i need need to collect the cells, centrifuge and transfer to HT medium. how many microlitre of HT medium should i use.
Thao Le
thank you for your suggestion. did i can know what type of supplements i have to use. could you share the supplement you are using.
Anton Shevyakov
thank you for your suggestion. i am planning to change my FBS.
Jody D Berry
thank you for your suggestion, did i can know what type of Hybridoma cloning factor i should use.
Hi colleague! Centrifugation, no way! I suggest you, in a 96 wells plate to absorb all the HAT media without disturbing the cells in the bottom of the plate. Next add supplemented HT media.
Do you have any other growth factors in the media? Hybridoma cloning factor?
You could just keep it in HAt as an option.
You could try adding a HCF or more higher % FBS
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