Question:
We have been using your human fab-pHast (PH-01) product for a while now and are very happy with the results in our imaging applications (we have seen signal to noise of over 70k). However I have recently tried running a black bottom plate on a plate reader, same set up, and am getting a high background signal. My signal to noise is about 1.5 in the best case scenario. Could you please advise how to increase the signal to noise in the plate reader format using fab-pHast?
Answer:
We recommend washing the cells with PBS before reading on the plate reader to help reduce background signal. You can find an internalization assay protocol here.
Question:
I’m using complete media (phenol red DMEM with 10% FBS) for my experiments, could this be contributing to background signal?
Answer:
Using phenol red-containing media could contribute to background signal. Replacing the media with PBS should reduce background significantly.
Question:
Do you have any recommendations on whether to use black opaque-bottom plates, black clear-bottom plates, or standard clear TC plates for maximal S/N?
Answer:
Clear-bottom plates offer the advantage of imaging, but they may introduce more background noise. If imaging is a priority, you might need to balance between signal strength and versatility.