碘化丙啶说明书

Propidium Iodide Nucleic Acid Stain

P-1304P-3566

propidium iodide

propidium iodide, 1 mg/mL solution in water

Quick FactsStorage upon receipt:󰂕󰂕Solid PI (P-1304), room temperature󰂕Solution of PI (P-3566), 4°CProtect from lightAbs/Em: 535/617 nm, bound to nucleic acidsIntroduction

Propidium iodide (PI) binds to DNA by intercalating betweenthe bases with little or no sequence preference and with a stoichi-ometry of one dye per 4–5 base pairs of DNA.1 PI also binds toRNA, necessitating treatment with nucleases to distinguish be-tween RNA and DNA staining. Once the dye is bound to nucleicacids, its fluorescence is enhanced 20- to 30-fold, the fluores-cence excitation maximum is shifted ~30–40 nm to the red andthe fluorescence emission maximum is shifted ~15 nm to theblue.2 Although its molar absorptivity (extinction coefficient) isrelatively low, PI exhibits a sufficiently large Stokes shift toallow simultaneous detection of nuclear DNA and fluorescein-labeled antibodies, provided the proper optical filters are used.Propidium iodide is suitable for fluorescence microscopy, confo-cal laser scanning microscopy, flow cytometry and fluorometry.PI is membrane impermeant and generally excluded fromviable cells. PI is commonly used for identifying dead cells in a

Figure 1. Absorption and fluorescence emission profiles of propidiumiodide bound to dsDNA.

MP 01304population and as a counterstain in multicolor fluorescent tech-niques. The counterstaining protocols below are compatible witha wide range of cytological labeling techniques — direct or indi-rect antibody-based detection methods, mRNA in situ hybridiza-tion or staining with fluorescent reagents specific for cellularstructures. These protocols can be modified for tissue staining.

Materials

Storage and Handling

Upon receipt, store the solid (P-1304) at room temperature,protected from light. The solid should be stable for at least ayear. Store the solution of PI (P-3566) at 4°C, protected fromlight. To make a stock solution from the solid form, dissolve PI(MW = 668.4) in deionized water (dH2O) at 1 mg/mL (1.5 mM)and store at 4°C, protected from light. When handled properly,solutions are stable for at least 6 months.

Caution: PI is a known mutagen. Solutions containing PIshould be poured through activated charcoal before disposal.The charcoal must then be incinerated to destroy the dye.

Fluorescence Spectral Characteristics

When bound to nucleic acids, the absorption maximum forPI is 535 nm and the fluorescence emission maximum is 617 nm(Figure 1). PI can be excited with a xenon or mercury-arc lampor with the 488 line of an argon-ion laser. Molecular Probesoffers high quality Omega Optical filter sets for fluorescencemicroscopy. Filter sets O-5725 and O-5729 are optimized tomatch the spectral properties of PI. Generally, PI fluorescence isdetected in the FL2 channel of flow cytometers.

Protocol for Counterstaining Adherent Cellsfor Fluorescence Microscopy

Sample Preparation

Use the fixation protocol appropriate for your sample. PI stain-ing is normally performed after all other staining. Note that per-meabilization of the cells is required for counterstaining with PI.

RNase Treatment

RNase treatment is required if samples are fixed in paraform-aldehyde, formaldehyde or glutaraldehyde. If samples are fixedwith methanol/acetic acid or acetone, RNase treatment is usuallynot required.

1.1 Equilibrate the sample briefly in 2X SSC (0.3 M NaCl,0.03 M sodium citrate, pH 7.0).

Propidium Iodide Nucleic Acid Stain

1.2 Incubate in 100 µg/mL DNase-free RNase in 2X SSC for20 minutes at 37°C.

1.3 Rinse samples 3 times, 1 minute each, in 2X SSC.

pH 7.4, 150 mM NaCl, 1 mM CaCl2, 0.5 mM MgCl2, 0.1%Nonidet® P-40). A 1 mL volume will be required for each cellsample.

4.2 Centrifuge the cell suspension from step 3.3, discard thesupernatant, tap to loosen the pellet and add 1 mL of PI-StainingBuffer. Incubate for 15 minutes at room temperature and analyzeby flow cytometry in the presence of the dye. If the cells are to beviewed by fluorescence microscopy, centrifuge the sample, re-move the supernatant and resuspend the cells in fresh buffer.Apply a drop of the suspension to a microscope slide, cover witha coverslip and view.

Counterstaining Protocol

2.1 Equilibrate the sample in 2X SSC.

2.2 Make a 500 nM solution of PI by diluting the 1 mg/mL(1.5 mM) stock solution 1:3000 in 2X SSC. About 300 µL isusually enough stain for one coverslip preparation. Incubatecells, covered with the dilute stain, for 1–5 minutes.

2.3 Rinse samples several times in 2X SSC. Drain excess bufferfrom coverslip and mount in a medium with an antifade reagentsuch as provided in Molecular Probes’ SlowFade® Antifade Kit,SlowFade Light Antifade Kit or ProLong® Antifade Kit.2.4 View sample using a fluorescence microscope with appropri-ate filters (see Fluorescence Spectral Characteristics).

Protocol for Chromosome FISH Counterstaining

Prepare the specimen according to standard procedures.3,4Briefly rinse the final preparations in dH2Obefore counter-

staining to remove residual buffer salts from the slide. Air dry.This final rinse will help reduce nonspecific background stainingon the glass.

Sample Preparation

Protocol for Counterstaining Cells in Suspensionfor Flow Cytometry

Sample Preparation

Use the fixation protocol appropriate for your sample, or usethe following protocol.

Counterstaining Protocol

3.1 Collect a volume of cell suspension corresponding to 2 × 105to 1 × 106 cells. Pellet the cells by centrifugation. Discard thesupernatant, tap the tube to resuspend pellet in the residual liquidand add 1 mL of phosphate-buffered saline (PBS) at room tem-perature.

3.2 Transfer the full volume of resuspended cells to 4 mL of

absolute ethanol at -20°C by pipetting the cell suspension slowlyinto the ethanol while vortexing at top speed. Leave in ethanol at-20°C for 5 to 15 minutes.

3.3 Pellet the cells by centrifugation, discard the ethanol, tap thetube to loosen the pellet and add 5 mL PBS at room temperature.Allow cells to rehydrate for 15 minutes.

5.1 Make a 1.5 µM PI staining solution by diluting the 1 mg/mL(1.5 mM) stock solution 1:1000 in PBS. Pipet 300 µL of thisstaining solution directly onto the specimen. If necessary, RNaseA (freshly made) may be added to a final concentration of

10 µg/mL. A plastic coverslip can be used to distribute the dyeevenly on the slide.

5.2 Incubate the specimen in the dark for 30 minutes at roomtemperature, or at 37°C if RNase is included.

5.3 Remove the coverslip and rinse briefly with PBS or dH2O toremove unbound dye.

5.4 Remove excess liquid from the slide by gently blotting

around the sample with an absorbent tissue. Place a glass cover-slip on the slide, and seal the edges with wax or nail polish.Alternatively, the preparation can be mounted in an antifade re-agent according to the manufacturer’s directions.

5.5 View sample using a fluorescence microscope with appropri-ate filters (see Fluorescence Spectral Characteristics).

Counterstaining Protocol

4.1 Make a 3 µM solution of PI by diluting the 1 mg/mL

(1.5 mM) stock solution 1:500 in Staining Buffer (100 mM Tris,

References

1. J Mol Biol 13, 269 (1965); 2. Meth Cell Biol 30, 417 (1989); 3. Meth Enzymol 168, 741 (1989); 4. Pardue, M.L. in Nucleic Acid Hybridization, A PracticalApproach,. B.D. Hames and S.J. Higgins, Eds., IRL Press, Oxford, England (1985).

Product List Current prices may be obtained from our Web site or from our Customer Service Department.

Cat #P-7481P-1304P-3566S-2828S-7461

Product Name

ProLong® Antifade Kit................................................................................................................................................................propidium iodide........................................................................................................................................................................propidium iodide *1.0 mg/mL solution in water*........................................................................................................................SlowFade® Antifade Kit.............................................................................................................................................................SlowFade® Light Antifade Kit.....................................................................................................................................................

Unit Size1 kit100 mg10 mL1 kit1 kit

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