I'd like to receive some input from people who worked with hybridomas. I need to resurrect them from being stored at -70C for about 5 years. I've never worked with hybridomas before and would appreciate any suggestions on what would be good growth factors, conditions and/or techniques.
Should be fairly simple. Normally you would just thaw them like any other cell line and put them into RPMI1640 + 10% FBS + non-essential amino acids + 2-mercaptoethanol (for better antibody quality, not needed during simple growth) -> look for recipes ar Invitrogen's or PAA's websites. Expensive media without sera or even w/o proteins are usually not necessary for a research lab. Cells can be adherent but only loosely attach. You should be able to suspend them by shaking the bottle. Usually cells should be split every 2-3 days. Medium colur is a good indicator for splitting time, too. Check the supernatant by dot blot for presence of the antibody and/or a functional assay with the corresponding antigen.
Hello kait,
you said that hybridoma have been stored in -70 C for 5 years !!!?
I think this cell will have low viability,because of long freezing time in -70(liquid nitrogen is better for long time freezing). therefor,its better to use medium with 20% FBS for first culture after thawing.
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