DNA can be stained by fluorescent dye. These DNA probes can stoichiometrically bind to DNA, which means the number of molecules of the probe bound is equivalent to the number of molecules of DNA found, so that the stained DNA can be quantitatively detected by flow cytometry due to its fluorescence.
The most widely used fluorescent dye is propidium iodide (PI), which has red fluorescence and can be excited at 488 nm. PI binds to DNA by intercalating between the bases with little or no sequence preference and with a stoichiometry of one dye per 4–5 base pairs of DNA. It can not only bind to double-strand DNA, but also double-strand RNA. So RNAase should be added before staining. Otherwise, the dye is excluded by the plasma membrane so that the cells have to be fixed or permeabilised before adding the dye.
Other fluorescent dye have also been used in DNA content analysis. 4',6-diamidino-2-phenylindole (DAPI) can binds strongly to A-T rich regions in DNA and it can pass through an intact cell membrane therefore it can be used to stain both live and fixed cells. Besides, hoechst33342, acridine orange, chromomycin A3, 7-aminoactinomycin D and ethidium bromide can be used for this purpose, too.
Since the data obtained is not a direct measure of cellular DNA content, reference cells with various amounts of DNA should be included in order to identify the position of the cells with the normal diploid amount of DNA. Some of the common reference cells often used for DNA measurements are human leukocytes or red blood cellsfrom chicken and trout.
1. Suspend approx 106 cells in 0.5 mL of PBS. Vortex gently (approx 5 s) or gently aspirate several times with a Pasteur pipet to obtain a mono-dispersed cell sus-pension, with minimal cell aggregation.
2. Fix cells by transferring this suspension, with a Pasteur pipet, into centrifuge tubes containing 4.5 mL of 70% ethanol, on ice. Keep cells in ethanol for at least 2 h at 4°C. Cells may be stored in 70% ethanol at 4°C for weeks.
3. Centrifuge the ethanol-suspended cells for 5 min at 300g. Decant ethanol thoroughly.
4. Suspend the cell pellet in 5 mL of PBS, wait approx 30 s and centrifuge at 300g for 5 min.
5. Suspend the cell pellet in 1 mL of PI staining solution. Keep in the dark at room temperature for 30 min, or at 37°C for 10 min.
6.Transfer sample to the flow cytometer and measure cell fluorescence. Maximum excitation of PI bound to DNA is at 536 nm, and emission is at 617 nm. Blue (488 nm) or green light lines of lasers are optimal for excitation of PI fluorescence.
1. Suspend the cell pellet in 1 mL of DAPI staining solution. Keep in the dark, at room temperature, for 10 min.
2. Transfer the sample to the flow cytometer and measure cell fluorescence. Maximum excitation of DAPI bound to DNA is at 359 nm, and emission is at 461 nm. For fluorescence excitation, use the available UV light laser line at the wavelength nearest to 359 nm. When a mercury arc lamp serves as the excitation source, use a UG1 excitation filter. A combination of appropriate dichroic mirrors and emission filters should be used to measure cell fluorescence at wave-lengths between 450 and 500 nm.