I have an in trans assay where I take conditioned media from transfected 293T cells and treat MG-63 cells with the conditioned media.

The problem I'm having is three of my controls (untransfected conditioned media, serum-free media (DMEM), and vector control have increased levels of phosphorylated activity in some experiments.

I serum starved my MG-63 cells for 24hrs prior to treatment. I also treat my cells with the conditioned at room temperature instead of 37C for 2hrs.

I'd like to know if serum starvation (0% serum) for 24hrs is too long and also if room temperature treatment with soluble protein is too harsh for the cell.

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