| |||||||||
| Login :: Register :: Search forums :: Top Users :: Reagent |
|
Position: Discussion forums > Bioscience biotechniques discussion forums > Cell biology and histology methods forum
|
cultivation of hybridomas
Posted by: Anouk (IP Hidden, New member, 1)
Date: April 25, 2007 08:01AM
hi!
I have serious problems to cultivate a hybridoma cell line. does anybody have experience with that? in my hands, the cells grow very slowly and reach a maximum of 3-4 x 10 to the 5 per ml. They are in suspension. I read, cells should be adherent...is that right? and how should the flasks be positioned? should they lie or stand within the incubator? are there any cytocines to add? I use simple RPMI medium with 10% FCS and the standard supplemention (L-Glutamine, Gantamycine, beta-mercaptoethanol)...is there something else to add? thank you very much! Anouk
Re: cultivation of hybridomas
Posted by: MDErin (IP Hidden, New member, 2)
Date: July 31, 2007 07:52AM
Adherent cell lines should always lie on their side, usually in a T75; use a T25 if they're not very confluent.
Re: cultivation of hybridomas
Posted by: aphio-igor (IP Hidden, New member, 6)
Date: April 2, 2008 02:15PM
It depends on the hybridoma, but most that I've dealt with are in suspension. As a general 'most are this way', your target range of concentration is 2e5 to 2e6 cells per mL, with most lines preferring 3e5 to 1e6. As for the media choice, again, it depends on the cells themselves, but most Balb/c based hybridomas I've worked with or developed have preferred DMEM as their basal constituent, adding 3% "LNN" (that is, 1% each of: 200mM L-Glutamine, MEM Non- essential Amino Acids, and NaPyruvate) per litre. 10% FBS is standard, and sometimes gentamycin is used as an antibiotic/ antimycotic (personal preference is to omit it, as it allows contaminant detection and containment as soon as it happens).
Again, that's the case for **most** hybridoma lines, and shouldn't be used exclusively as a hard and fast rule. If you purchased the cells from a provider, might I ask what the cell line's name is? I might be able to provide more help with that. Beyond that, I'll restate what was said before, in that the T- flasks should have a slanted neck leading to the cap. You want the flask's two largest sides laid parallel to to the incubator's shelf, with the neck slanted slightly up. If the cap has vents for the gas exchange for CO2, you're fine, but if not, don't remember to loosen the cap a tad once in the incubator. I hope that this helps, and feel free to ask any questions... it may take a while to reply, as I have a newborn at home and I'm the "official diaper changer" ;) Kind regards, Pete Gramp (aphio-igor)
|
We are moving ... Please post to our new community forums
|
|
||||||||