CELL CYCLE ANALYSIS PROPIDIUM IODIDE ACRIDINE ORANGE HOECHST/DAPI PROPIDIUM IODIDE: The most commonly used dye for DNA content/cell cycle analysis is PROPIDIUM IODIDE (PI). It can be used to stain whole cells or isolated nuclei. The PI intercalates into the major groove of double-stranded DNA and produces a highly fluorescent adduct that can be excited at 488 nm with a broad emission centred around 600 nm. Since PI can also bind to double-stranded RNA, it is necessary to treat the cells with RNase for optimal DNA resolution. The excitation of PI at 488 nm facilitates its use on the benchtop cytomters. However, PI can also be excited in the U.V. (351-364 nm line from the argon laser) which should be considered when performing multicolour analysis on the multibeam cell sorters. Protocol for staining whole cells with PI: 1. Harvest cells and prepare single cell suspension in buffer (e.g. PBS + 2% FBS; PBS + 0.1% BSA) 2. Wash cells X2 and resuspend at 1-2 x 106 cells/ml. 3. Aliquot 1 ml cells in a 15 ml polypropylene, V-bottomed tube and add 3 ml cold absolute ethanol. (The ethanol can be added forcibly by expelling from a pipette or dropwise while vortexing…determine the best method for each cell type to prevent clumping and cell loss.) 4. Fix cells for at least 1 hour at 4oC. (Cells may be stored in 70 % ethanol at -20 oC for several weeks prior to PI staining and flow cytometric analysis). 5. Wash cells X2 in PBS. (It may be necessary to centrifuge cells at a slightly higher "g" to pellet after ethanol fixation as the cells become floculent.) 6. Add 1 ml of propidium iodide staining solution to cell pellet and mix well. Add 50 ul of RNase A stock solution and incubate 3 hr at 4oC. Final Concentration 0.5ug/ml. 7. Store samples at 4oC until analysed by flow cytometry. top of page REAGENTS: Propidium Iodide Staining Solution: 3.8 mM sodium citrate, 50 ug/ml PI [Sigma, P 4170] in PBS. RNase A stock solution: 10 ug/ml RNase A [Worthington Biochemicals, RASE LS005649, LS005650] (boiled for 5 min, aliquoted and stored frozen at -20oC). References: Crissman HA, Steinkamp JA. Rapid simultaneous measurement of DNA, protein and cell volume in single cells from large mammalian cell populations. J. Cell Biol., 59:766, 1973. Krishan A. Rapid flow cytofluorometric analysis of cell cycle by propidium iodide staining. J. Cell Biol., 66:188, 1975. ACRIDINE ORANGE: Differential Staining of DNA/RNA with ACRIDINE ORANGE (AO) can be performed for simultaneous assessment of DNA and RNA content of cells. The AO exhibits different spectral characteristics when bound to DNA or RNA that can be excited at 488 nm with either a GREEN or RED fluorescence respectively. Protocol for staining whole cells with AO: 1. Wash cells X2 in buffer (e.g. PBS + 2% FBS; PBS + 0.1% BSA). 2. Resuspend cells to 1 x 106 cells/ml in buffer. 3. Add 200 ml to a clean test tube and add 400 ml of Solution A. Mix gently by swirling tube.(DO NOT VORTEX). 4. Stand for 15 seconds and then add 1.2 ml of ice-cold Solution B. 5. Stand on ice for 5 to 15 min to allow sample to equilibrate and analyze for fluorescence immediately. top of page REAGENTS: Solution A: 0.1 % Triton X-100, 0.08N HCl, 0.15M NaCl Solution B: 6 ug/ml Acridine Orange [Sigma, A 6014], 1 mM EDTA-Na, 0.15M NaCl, 0.2M NaPO4, 0.1M Citric acid buffer, pH 6.0 References: Traganos F, Darzynkiewicz Z, Sharpless T et al. Simultaneous staining of ribonucleic and deoxyribonucleic acids in unfixed cells using acridine orange in a flow cytofluorometric system. J. Histochem. Cytochem., 25:46, 1977. Darzynkiewicz Z, Traganos F, Melamed MR. New cell cycle compartments identified by multiparameter flow cytometry. Cytometry, 1:98, 1980. HOECHST: The Hoechst dyes 33342 and 33258 are bis-benzimide derivatives. They can be used for cell cycle analysis of viable cells and can be used at low concentrations limiting toxicity problems. The Hoechst dyes bind to AT-rich regions of the DNA and when excited with a U.V. source (e.g. . (351-364 nm line from the argon laser) produce bright fluorescence at 465 nm. DAPI (diamidino-2-phenylindole 2HCl) is also an AT binder with similar spectral properties to the Hoechst dyes. Protocol for staining whole cells with HOECHST: 1.Wash cells X2 in buffer (e.g. PBS + 2% FBS; PBS + 0.1% BSA). 2. Resuspend cells to 1 x 106 cells/ml in buffer. 3. Add an equal volume of Hoechst dye (working solution 5 mM). 4. Incubate for 15 min at 37oC. 5. Analyse for fluorescence or cells can be washed and fixed in paraformaldehyde (1% in PBS) for future analysis. top of page REAGENTS: Stock Solution: Dissolve 2mg of Hoechst 33342 [vendor, cat no.] in 1 ml of DMSO. Store at 4oC. Working Solution: Add 1.4 ml of stock solution to 1 ml PBS. References: Loewe H, Urbanietz J. Arnzeimforsch, 24:1927, 1974. Latt SA. Microfluorometric detection of deoxyribonucleic acid replication in human metaphase chromosomes. Proc. Natl. Acad. Sci. USA. 70:3395, 1973. Latt SA, Stetten G. Spectral studies on 33258 Hoechst and related bisbenzimidazole dyes useful for fluorescent detection of deoxyribonucleic acid synthesis. J. Histochem. Cytochem., 24:24, 1976. Bigler RD. A comparison of low-power helium-cadmium and argon ultraviolet lasers in commercial flow cytometers. Cytometry, 8:441, 1987. Shapiro HM. Flow cytometric estimation of DNA and RNA content in intact cells stained with Hoechst 33342 and pyronin Y. Cytometry, 2:143, 1981.