Hi Sara

You have not indicated the passage number for your cell line: could it be that your passage number is too high? Or that your splitting has been done past-confluency too often?

I have never noticed crystal-like structures with T-REx cells: you said your cells are modified with channels? Could they be exporting something that precipitates in the medium?

The other annoying possibility is mycoplasma contamination: maybe worth testing for it.

Hello Sara.

If you did not have any problems during last several months, and now you find the problem? Or you already had problems and now you realized? Give more detail. I did not have any work experience with the culture bottle. Everyone I know around uses single culture dish or multiple dishes together like 6-wells or 24-well dish.

It sounds your cells may be contaminated and you need a new stock of HEK293T cells. Get a new stock. I also suggest that CO2 incubator needs cleaning and sterilization, too. It is the warm bed where microbes can sit, proliferate and make particles. They can travel around by the light weights and fanning movements all the year round. In fact, the particle gets through the gaps between the loosened lid and bottle, round culture dish lid and bottom. Even the medium move around by the fanning circulation movements. Change your Rex culture container system, it is untouchable even if you found something wrong, it only extends the area exposed by transfer procedure to be easily contaminated by the ever-so-whirling CO2 incubator.

Regards.

Thanks alot for your answer

INCUBATOR is OK

but one cell line dont groqww ell.. intial passage number was 8 and now is 20 . Maybe its related to numbers.