Analyze on flow cytometer with excitation at 488 nm and emission collected at >550 nm.
PI is easily excited at 488 nm. The dye has a broad fluorescence emission and can be detected with photomultiplier tubes (PMTs) normally used for phycoerythrin (∼585 nm) or at longer wavelengths (≥650 nm).
Amplify the PMT signal logarithmically to distinguish populations of permeable (and presumed dead) cells from viable cells. Adjust the PMT high voltage such that bright cells are well separated from dim, viable cells. It is often easy to identify nonviable cells on a bivariate plot of forward light scatter (see Fig. 9.2.1).
Figure 9.2.1. Identification of nonviable cells with propidium iodide (PI). Nonviable cells are more than two decades brighter than the unstained, viable cells. Gating on a one-parameter histogram is sufficient to identify the viable population. Region 1: viable cells; region 2: nonviable cells.
Dead cells can be live-gated, but unless one is absolutely sure of the viable population, it is always much better to collect ungated listmode data and perform gating after the raw data files are collected. Populations that may not be obvious on the flow cytometer display will be seen during subsequent data analysis. Moreover, any gating can be done and redone without losing cells.