I am culturing N2A cells with stable transfection of Mock, hmWT PS1 and mutated PS1 (E280A). They all express antibiotic resistance and are grown in DMEM + 10% FBS and 0.4% Zeocin. My hmWT cell line is having some problem while the other two seem quite fine. They stop attaching, and after changing media every two days, they appear to have many dead cells just floating in the media. The cells that are attached display some odd morphology, and seem like they started differentiating. Any advice on what to do? Maybe I should grow them without antibiotic for some time?
Are you sure that your cells carry again your transgene? It seems that they loose their resistance and dying because you select zeocin resistant cells. Are you sure of your cells and vector for this cell line ?
When viewed under the microscope, are you seeing black, granule like deposits only on the cells that are dying ? If this is the case, it could be just that the cells are contaminated with mycoplasma.
Ideally start with a fresh batch of cells from reliable source after cleaning the incubator thoroughly.
Biju Joseph
Did you switch batches of medium at the time they started dying? I have had this happen with J774A.1 cells. (And invisible contamination is also a possibility, as noted above.)
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