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.
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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).
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,
Krishan A. Rapid flow cytofluorometric analysis of cell cycle by propidium iodide
staining. J. Cell Biol., 66:188, 1975.
(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
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.
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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
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.
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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.
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.