Propidium iodide color interference - (Apr/12/2012 )
I'm running an Annexin V/PI apoptosis assay, and I'd like to be able to discriminate T cell types in the assay. I'm worried that PI, with its ludicrously broad emission and excitation peaks, is going to screw with my other colors. Does anyone have insight into what dyes can be safely run with PI?
Right now I'm using PE, PE-Cy5 (yes I know this interferes), APC, APC-Cy7, and I'm thinking of adding PE-Cy7, DyLight 405, and Pacific Orange, but I also have a PE-Cy5.5, which I suspect can be safely used with PI. Our LSRII also has a green laser, which I think is 543nm, in addition to the 488nm, 405nm, and 633nm.
annexin V/PI method is very popular. PI shoudln't be a problem. In fact I only stained with PI in many of my experiments. Depends if you want late apoptosis or early apoptosis. I found SSC-A and PE-Cy7 better than other filters. I also used logarithmic PE-Cy7, but SSC-A was normal.
Yes, PI is popular, but most papers don't use it in conjunction with other cell markers. I'd like to be able to distinguish between single positive, double positive, double negative, and gamma delta thymocytes, so I need at least four colors that aren't FITC or overlapping with PI.
PE-Cy7 is my favorite conjugate, there's no overlap with any other dye, plus it's surprisingly bright.
They are also some alternative marker for necrosis which can replace PI that might be a way to explore
They are a similar discussion over here