B cells cultures. Immunoglobuline production - (Jul/07/2013 )

Hi,

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.

Thank you,

YM

I don't do this experiment, but I think day 14 is too late. Last time I did plaque assay and kept cells in incubator max 7 days. Can you add more media if the wells are still not filled and continue till day 14?