Fundamental study for flow cytometric DNA analysis using propidium iodide

Abstract

It is essential to fully understand the chemical mechanisms of dye-binding reaction of DNA in order to obtain reliable results in flow cytometric (FCM) analysis of cellular DNA content. There are few reports on the binding process of propidium iodide (PI) to DNA although PI is frequently used as fluorescent probe for DNA analysis. The binding mechanisms was investigated with (fluorescent) spectrophotometer and flow cytometer using calf thymus DNA and Syrian hamster spleen cells Moreover, optimal concentration of PI for DNA analysis was determined and effects of RNAse treatment was examined.
The absorption maximum was shifted from 494 nm to 540 nm in PI-DNA complex, and a well-defined isosbetic point was demonstrated at 525 nm. Excitation at 488 nm, where the relative quantum yield of PI bound to DNA was 24 times greater than that of free dye, results in an emission maximum at about 605 nm. Two modes of interaction were distinguished between PI and DNA, i. e., complex I which was observed only on small values of DNA/PI (K.=3×10⁷/M) and complex II (K=8×10⁵/M). Association constant (K) of hamster spleen cells was estimated to be 3×10⁶/M from the FCM data. Optimal concentration of PI for cell staining was defined as 50 μg/ml, and in this concentration binding sites on nuclear DNA could be saturated by PI molecules. The channel of G1/GO peak of DNA histogram was greatly varied by slight changes of PI concentration when cells were stained with lower dye concentrations. RNAse treatment did not influence the position of G1/G0 peak, while it slightly improved CV (coefficient of variation) values of the peak in DNA histograms.