I wish to perform DCFDA assay (Sigma) on primary microglia after treatment. The number of cells is so low that I cannot proceed for Flow-based analysis techniques. Therefore, I am thinking of visualizing and counting the cells expressing green fluorescent DCF. I wish to express it as a percentage of total cell count, done by Hoechst dye staining of the nucleus of all the cells. Is it possible to do so?
I think it is possible to use a combination of Hoechst dye and DCFDA in your case.
For hoechst, yes you can easily quantify manually the number of apoptotic nuclei. However, it is not appropriate to do so for dcfh-da. It is because, ROS are found scattered throughout the cell body, and some part of one cell might be stained brighter than the other parts. That is why for dcfh-da you need to measure the fluorescence intensity.
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