B cell culture. Question!!
I am new in the field of cell culture. I am performing an experiment to detect IgE production in the supernatants of isolated and stimulated B lymphocytes. According to my protocol I should take the supernatants from my cell culture (I plate 100.000 B cells in 100 microlitres in 96 well plate) at day 14 in order to analyze the IgE. But at that day the medium is completely yellow.
Does anybody have any experience on measuring IgE in B cell culture supernatants? Is day 14 already too late? The medium is yellow so, I am afraid most of the cells are already dead at that time.
I would appreciate you share your experiences with me.
Re: B cell culture. Question!!
Protocols that take many days of culture usually need at least one change of media at some point of the culture in order to keep the cells properly "fed".
In protocols that involve Ab isotype change the media is replaced less times than in other protocols, or so I have read.
I do not have experience with IgE but for IgG I believe you need a minimum of 7 days of incubation. I suggest you look for info regarding your experiment in order to check when you have to replace media, how many days you need to culture B cells to have measurable titles of IgE, etc.
If you have access to Current Protocols in Immunology I suggest you check it. Good luck
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