This Sample Unit contains the full text of the published Unit, including expert commentary sections with critical information designed to ensure the success of your experiments. 

UNIT 7.3

Differential Staining of DNA and RNA

Analysis of nucleic acids is a common application of flow cytometry (reviewed in UNIT 7.1). Measurement of the DNA content of cells allows investigators to determine DNA ploidy (UNIT 7.5) as well as to analyze populations of cells in various phases of the cell cycle. Such studies, along with analysis of DNA strand breaks to detect apoptotic cells (UNIT 7.4), are increasingly important in disease diagnosis. Cell cycle analysis may also be done by means of differential staining of DNA and RNA, as described in this unit. Determining the RNA content allows one to discriminate G₀ versus G₁ cells and detect cell differentiation.

Differential staining of DNA and RNA can be accomplished using either the metachromatic dye acridine orange (AO) or a combination of pyronin Y (PY) and Hoechst 33342 (PY-Hoechst 33342). Acridine orange is a versatile dye that can be used to stain a variety of different constituents in cells (Darzynkiewicz and Kapuscinski, 1990). Following cell fixation and removal of RNA with RNase, AO can also be used for differential staining of double-stranded versus single-stranded DNA sections to analyze the sensitivity of DNA to denaturation.

The mechanisms of interaction with nucleic acids, and in particular the spectral changes on binding to DNA or RNA, are very much different for AO and PY-Hoechst 33342 (Table 7.3.1). Dye binding characteristics and specific features of the method based on use of AO (see Basic Protocol 1 and Alternate Protocol) and the method utilizing PY (see Basic Protocol 2) are therefore described separately. A procedure for determining the specificity of cell staining with AO or PY-Hoechst 33342 is also provided (see Support Protocol; also see UNIT 7.2 for more information on quality control in nucleic acid analysis).

BASIC PROTOCOL 1

DIFFERENTIAL STAINING OF DNA AND RNA OF UNFIXED CELLS WITH ACRIDINE ORANGE

Materials

Cells to be stained (APPENDIX 3B): 10⁶ cells/ml suspended in tissue culture medium containing 10% (v/v) serum or 1% (w/v) BSA

Cell permeabilizing solution (see recipe), ice cold

Acridine orange (AO) staining solution (see recipe), ice cold

Flow cytometer equipped either with a 488- or 457-nm argon ion laser (or both lasers) or with a mercury arc lamp

1. Set up the flow cytometer with excitation at 488 nm, using emission filters and a dichroic mirror that discriminate green fluorescence (measured at 530±15 nm) and red luminescence (measured preferably above 640 or 650 nm).

 
Maximum absorption by AO occurs at ~455 to 490 nm. The 488-nm line of the argon ion laser is the most commonly used excitation wavelength. In instruments having a mercury or xenon lamp, blue excitation filters can be used (for example, a BG 12 short-pass filter transmitting below 470 nm or band-pass combination fitters transmitting between 460 and 500 may be used). Optimal excitation can be achieved using two lasers, one tuned to 488 nm (DNA detection, green fluorescence) and another to 457 nm (RNA detection, red luminescence).
 
2. Transfer a 0.2-ml aliquot of the original cell suspension to a small glass or plastic tube (e.g., 2- or 5-ml volume). Chill on ice.

 
The 0.2-ml aliquot should have 2×10⁵ cells suspended in tissue culture medium containing 10% (v/v) serum or 1% (w/v) BSA. The serum or BSA protects cells from lysis by detergent in step 3.
 
3. Gently add 0.4 ml ice-cold cell permeabilizing solution. Wait 15 sec, keeping cells on ice.

 
4. Gently add 1.2 ml ice-cold AO staining solution. Keep cells on ice in the dark.

 
5. Measure and record cell fluorescence in the flow cytometer during the 2 to 10 min after addition of AO staining solution.

 
The sample should be kept on ice prior to and during the measurement. Vortexing or syringing cells in the permeabilizing solution, especially in the absence of any serum or proteins in the original cell suspension, results in disintegration of plasma membranes and isolation of cell nuclei. The RNA content of isolated nuclei, therefore, can be measured after plasma membrane disruption in this way. Visual inspection of the nuclei under phase-contrast or UV light microscopy is essential to estimate the efficiency of the isolation, which can be controlled by selecting either an optimal time and speed of vortexing or an optimal number of syringings.

DNA frequency histograms (green fluorescence) can be deconvoluted to obtain the proportion of cells in G₁ versus S versus G₂/M (see UNIT 7.5).
 

ALTERNATE PROTOCOL

DIFFERENTIAL STAINING OF FIXED CELLS WITH ACRIDINE ORANGE

Cells to be stained

PBS (APPENDIX 2A), ice cold

70% ethanol, ice cold

Centrifuge, 4°C

Additional reagents and equipment for trypsinizing adherent cells (UNIT 5.2 or APPENDIX 3B) or dissociating cells from tissues (UNIT 5.2)

1. a. For cells in suspension culture or hematologic samples: Rinse cells once with ice-cold PBS and suspend in ice-cold PBS at ~10⁶ cells/ml.

 

1. b. For cells attached to tissue culture plates: Collect cells from flasks or petri plates by trypsinization, pool the trypsinized cells with cells floating in the medium (mostly detached mitotic and dead cells), and rinse once with medium containing serum to inactivate the trypsin (see UNIT 5.2 or APPENDIX 3B for details of this procedure). Suspend cells in ice-cold PBS at ~10⁶ cells/ml.

 
Other means of trypsin inactivation such as addition of protease inhibitors may also be used.
 

1. c. For cells isolated from solid tumors: Rinse cells free of any enzyme used for cell dissociation and suspend in ice-cold PBS at ~10⁶ cells/ml.

 
The final cell suspension should be well dispersed (no aggregates), with a density 5 ×10⁶ cells/ml. The cells should not be stored on ice for longer than 30 min before fixation.
 
2. With a Pasteur pipet transfer 1 ml cell suspension to a 15-ml conical glass tube containing 10 ml ice-cold 70% ethanol. Fix cells 32 hr on ice.

 
To minimize cell clumping, rapidly injecting the cell suspension into the fixative, rather than layering onto the surface and then mixing, is preferred. The reverse order (i.e., addition of ethanol to cell suspensions) results in more extensive cell loss due to cell adherence to the glass surface and aggregation. The time of fixation in ethanol may vary, but at least 2 hr should be allowed for cells to be fixed. Cells may also be stored in ethanol at 4°C for several months.
 
3. Centrifuge tubes 5 min at 300 ' g, 4°C. Remove all ethanol, rinse cells once with ice-cold PBS, and suspend in ice-cold PBS at a density of <2 ×10⁶ cells/ml.

 
4. Withdraw 0.2 ml cell suspension (2×10⁵ cells) and transfer to a small tube (e.g., 2 or 5 ml volume). Chill on ice.

 
5. Add 0.4 ml ice-cold permeabilizing solution. Wait 15 sec, keeping cells on ice.

 
6. Add 1.2 ml ice-cold AO staining solution. Keep cells on ice.

 
Although the presence of Triton X-100 in the permeabilizing solution is not necessary in the case of fixed cells, it does not interfere with staining. Therefore, the same solution can be used in both this protocol and Basic Protocol 1.
 
7. Measure cell fluorescence as described for unfixed cells (see Basic Protocol 1, step 5).

 
To assess the contribution of RNA to the detected luminescence, following step 3 duplicate cell samples can be incubated with RNase A (100 µg/ml) for 30 min at 37°C, prior to staining with AO.
 

BASIC PROTOCOL 2

DIFFERENTIAL STAINING OF DNA AND RNA WITH HOECHST 33342 AND PYRONIN Y

In this procedure the cells are fixed in ethanol, then resuspended in a solution of Hoechst 33342 and PY and measured by flow cytometry while suspended in this solution. Because Hoechst 33342 is a more specific DNA fluorochrome than is AO, this protocol is preferred when more accurate DNA content measurements are desired.

Materials

Cells to be stained

PBS (APPENDIX 2A), ice cold

70% ethanol, ice cold

Hanks' balanced salt solution (HBSS) containing Mg²⁺ and Ca²⁺ (APPENDIX 2A),

ice cold

 

Pyronin Y (PY)-Hoechst 33342 staining solution (see recipe), ice cold

Centrifuge, 4°C

Flow cytometer equipped either with two lasers or with one laser and a mercury arc lamp

Additional reagents and equipment for trypsinizing adherent cells (UNIT 5.2 or APPENDIX 3B) or dissociating cells from tissues (UNIT 5.2)

1. a. For cells in suspension culture or hematologic samples: Rinse cells once with ice-cold PBS and suspend in ice-cold PBS at ~10⁶ cells/ml.

 

1. b. For cells attached to tissue culture plates: Collect cells from flasks or petri plates by trypsinization, pool the trypsinized cells with cells floating in the medium (mostly detached mitotic and dead cells), and rinse once with medium containing serum to inactivate the trypsin (see UNIT 5.2 or APPENDIX 3B for details of this procedure). Suspend cells in ice-cold PBS at ~10⁶ cells/ml.

 
Other means of trypsin inactivation such as addition of protease inhibitors may also be used.
 

1. c. For cells isolated from solid tumors: Rinse cells free of any enzyme used for cell dissociation and suspend in ice-cold PBS at ~10⁶ cells/ml.

 
The final cell suspension should be well dispersed (no aggregates) with a density no higher than 5× 10⁶ cells/ml. Do not store cells on ice longer than 30 min before fixation.
 
2. With a Pasteur pipet transfer 1 ml cell suspension to a 15-ml glass tube containing 10 ml ice-cold 70% ethanol. Fix cells for 32 hr.

 
To minimize cell clumping, rapidly injecting the cell suspension into the fixative, rather than layering onto the surface and then mixing, is preferable. The reverse order (i.e., addition of ethanol to cell suspensions) results in more extensive cell loss due to cell aggregation and adherence of cells to the glass surface. The time of fixation (storage) in ethanol may vary from 2 hr to several months at 4°C.
 
3. Centrifuge tubes 5 min at 300×g, 4 °C. Remove all ethanol, rinse cells once with ice-cold HBSS containing Mg²⁺ and Ca²⁺ , and suspend in ice-cold HBSS containing Mg²⁺ and Ca²⁺ and, at a density <2× 10⁶ cells/ml.

 
4. Mix 0.5 ml cell suspension with 0.5 ml ice-cold PY-Hoechst 33342 staining solution in a small tube. Keep sample in the dark.

 
5. Set up the two-laser flow cytometer with one laser providing excitation in the UV, and the other providing excitation in the blue or green. Alternatively, use a combination of a laser and mercury lamp. Collect blue Hoechst 33342 fluorescence (DNA) using a 480  15 nm band-pass filter and green PY fluorescence (RNA) with a 570- or 580-nm long-pass filter.

 
Maximal excitation of DNA-bound Hoechst 33342 fluorescence is at 350 nm; optimal excitation can be achieved with the 350/356-nm line of the krypton ion laser, the 351/363-nm line of the argon laser, or with UV filters (e.g., UG 1 short-pass filter transmitting below 390 nm) in a mercury lamp system. Optimal excitation of PY is in green light (550 nm), but PY can be excited with any wavelength between 488 and 530 nm, by several lines available in either krypton or argon ion lasers (Crissman et al., 1985). Maximal emission of the RNA-bound PY fluorescence is at 570 nm (see Table 7.3.1).
 
6. Measure cell fluorescence 20 min after addition of the PY-Hoechst 33342 staining solution.

 

SUPPORT PROTOCOL

DETERMINATION OF SPECIFICITY OF CELL STAINING

Cells fixed in ethanol (see Alternate Protocol, step 2, or see Basic Protocol 2, step 2)

RNase solution: 100 mg/ml RNase A in PBS containing Mg²⁺

DNase solution: 500 mg/ml DNase I in PBS containing Mg²⁺

1. a. To estimate the stainability of RNA: Centrifuge ethanol-fixed cells 5 min at 300×g, 4°C, thoroughly remove all ethanol by decanting, and resuspend the cell pellet (10⁶ cells) in 1 ml RNase solution. Incubate 30 min at 37°C.

 
Use DNase-free RNase. Otherwise, boil the RNase solution for 5 min and cool to 37°C before use.
 

1. b. To estimate the stainability of DNA: Centrifuge cells as in step 1a, but resuspend the cell pellet in 1 ml DNase solution. Incubate 30 min at 37°C.

 
2. Stain cells with AO (see Alternate Protocol, steps 4 to 6) or with PY-Hoechst 33342 (see Basic Protocol 2, step 4).

 
For unfixed cells stained with AO (Basic Protocol 1), cell suspensions already treated with the permeabilizing and staining solutions (see Basic Protocol 1, steps 2 to 4) may be subsequently treated with 100 µg/ml of RNase A and incubated 30 min at 24°C prior to fluorescence measurements.
 
3. Perform flow cytometry and make appropriate measurements (see Basic Protocol 1, step 5, for AO; see Basic Protocol 2, steps 5 and 6, for PY-Hoechst 33342).

 

REAGENTS AND SOLUTIONS

Stock solution: Dissolve 1 mg/ml AO in distilled water and store 6 months in the dark at 4°C.

Staining solution

370 ml 0.1 M citric acid (37 mM final)

630 ml 0.2 M Na₂ HPO₄ (126 mM final)

8.77 g NaCl (150 mM final)

340 mg Na₂EDTA (1 mM final)

6.0 ml 1 mg/ml AO stock solution (see above; 6 mg/ml final)

H₂O to 1 liter

Store 6 months in dark or foil-wrapped bottles at 4°C

The quality of the AO is essential. Highly purified AO (mol. wt. 302) is available from Molecular Probes.

Stir to dissolve the sodium chloride before adding the EDTA. Continue stirring until completely dissolved. An equivalent amount of Na₄ EDTA may be used; H₄EDTA can also be used but will require a longer time to dissolve.

The AO staining solution may be kept in a brown-colored automatic-dispensing pipet bottle set at 1.2 ml.

1 ml Triton X-100 (0.1% final)

80 ml 1.0 M HCl (80 mM final)

8.77 g NaCl (150 mM final)

H₂O to 1 liter

Store  6 months at 4°C

The permeabilizing solution may be kept in an automatic-dispensing pipet bottle set at 0.4 ml.

2 mg Hoechst 33342

4 mg PY

Add HBSS containing Mg²⁺ and Ca²⁺ (APPENDIX 2A) to 1 liter

Prepare fresh

Hoechst 33342 (mol. wt. 652) is available from Molecular Probes. Pure PY is available from Polysciences. Most PY from other sources has 30% to 40% impurities and should be purified by chloroform extraction and recrystallization from methanol (Kapuscinski and Darzynkiewicz, 1987).

COMMENTARY

Background Information

Differential staining with AO

Interaction of AO with ss nucleic acids is a complex, multistep process initiated by AO intercalation between neighboring bases, neutralization of the polymer charge by the cationic dye, and subsequent condensation and agglomeration (precipitation; solute-to-solid state transition) of the product (Kapuscinski et al., 1982). The condensation reaction is highly cooperative. In the final product, AO molecules are interspaced with DNA bases, forming stacks of alternating dye-base composition, which by virtue of their solid-state form are protected, to a limited degree, from interaction with oxygen or water molecules. The absorption spectrum of AO in these precipitated products is blueshifted compared to that of the intercalated AO, with maximum absorption ranging between 426 and 458 nm, depending on the base composition of the nucleic acid. The emission of AO in these complexes also varies, between 630 and 644 nm, depending on the base composition. The lifetime of the red emission at room temperature is >20 nsec (Darzynkiewicz and Kapuscinski, 1990). These spectral properties of AO in complexes with ss nucleic acids are suggestive of intersystem crossing (phosphorescence; T₁ to S₀ transition) rather than fluorescence. The solid-state nature of the complexes, similar to freezing AO in solution, may facilitate the intersystem crossing by partially eliminating collision quenching, which otherwise occurs due to the long lifetime of the T₁ excited state in the presence of oxygen and solvents.

In the cell, large sections of rRNA and tRNA have ds conformation. Therefore, to obtain differential staining of DNA versus RNA with AO, these sections have to be selectively denatured, under conditions in which DNA still remains double stranded. This is accomplished by treatment of cells with AO in the presence of the chelating agent EDTA (Darzynkiewicz et al., 1976). By breaking RNA-protein interactions in ribosomes that stabilize ds RNA, EDTA promotes denaturation of ds RNA, which occurs as a result of interaction with AO. The RNA-selective denaturing properties of AO result from the fact that this ligand has higher affinity to ss RNA than ss DNA (Kapuscinski and Darzynkiewicz, 1989). The denaturation itself results from the fact that, at increased dye/phosphate (D/P) ratio, binding of AO to ss nucleic acids becomes thermodynamically preferable; the weaker but more numerous 1:1 (D/P) interactions of AO with ss sections thermodynamically dominate the stronger but fewer (1:4) sites of AO intercalation to ds regions (Kapuscinski and Darzynkiewicz, 1989). Thus, increasing D/P promotes denaturation of ds RNA sections.

Cell staining with AO is generally done in salt solutions of relatively high ionic strength (0.1 to 0.2 M NaCl). Because of the significant electrostatic component in binding of AO to nucleic acids, competitive interactions between the AO⁺ and Na⁺ result, and it is the concentration of free AO in solution (rather than absolute D/P calculated on the basis of molar ratios of the dye and nucleic acid in the sample) that is of importance for selective RNA denaturation (Darzynkiewicz and Kapuscinski, 1990).

To recapitulate, AO has two distinct functions in the mechanism of metachromatic staining of RNA, namely, it (1) denatures ds RNA and (2) differentially stains RNA (after its conversion to ss form) versus DNA. At a given ionic strength, selective RNA denaturation can be achieved only within a narrow concentration range of free dye. Too low an AO concentration produces incomplete RNA denaturation (both DNA and portions of RNA then stain green), whereas a too high concentration also leads to denaturation of DNA (both RNA and DNA stain red). Thus acridine orange, in contrast to most other dyes used in cytometry, requires very stringent conditions, especially with regard to concentration of AO and ionic strength of the staining solutions. The protocol of cell staining described in Basic Protocol 1 and Alternate Protocol has been established after testing a variety of ionic conditions, pH, and dye concentrations (Darzynkiewicz et al., 1976; Traganos et al., 1977).

Differential staining with PY-Hoechst 33342

1. Intercalary mode of binding. PY binds by intercalation to ds nucleic acids. Its binding affinity to ds RNA is severalfold higher than to ds DNA. In the intercalated form, whether bound to RNA or DNA, the dye has maximal absorption between 547 and 563 nm and fluoresces with maximum emission between 565 and 574 nm; the variation is due to differences in base composition of the nucleic acids (Kapuscinski and Darzynkiewicz, 1987). Its quantum yield also varies widely with changes in base composition. In contrast to AO, which stains total cellular RNA, PY used as fluorochrome can detect only ds sections of RNA and is sensitive to the AU/GC base ratio.

2. Condensation and precipitation of nucleic acids. Like AO binding, binding of PY to ss nucleic acids results in condensation (precipitation) of the product. The mechanism of condensation and the structure of the complexes are similar to those generated by AO. In contrast to AO, however, PY fluorescence is nearly totally quenched in these complexes (Kapuscinski and Darzynkiewicz, 1987). In absorption microscopy these products are characterized by lavender color.

3. Stoichiometry of RNA detection. The stoichiometry and thermodynamics of binding of PY to ds and ss nucleic acids are similar to those of AO. Thus, PY can denature the ds sections of nucleic acids, rendering them single-stranded and causing their condensation and agglomeration (Kapuscinski and Darzynkiewicz, 1987). At increasing PY concentration, therefore, the fluorescence of PY bound to ds RNA is suppressed because of the progressive denaturation of the ds sections ("self-extinguishing" effect of PY). Similarly, as in the case of AO, selective stainability of RNA with PY can be obtained at only a relatively narrow range of dye concentrations.

4. DNA stainability with PY. PY, having affinity to ds DNA, can also stain DNA. Its binding to DNA, however, can be suppressed by DNA-specific ligands such as methyl green and Hoechst 33342 (Tanke et al., 1980; Shapiro, 1981). In the presence of these dyes, therefore, PY can be used as a specific RNA fluorochrome. Dual cell staining with Hoechst 33342 and PY as described in Basic Protocol 2 provides the basis for simultaneous detection of DNA and RNA in flow cytometry (Shapiro, 1981; Darzynkiewicz et al., 1987).

Comparison of AO and PY-Hoechst 33342 methods

Quantitative aspect of the methods. The stoichiometry of RNA measurement is better assured by the AO methodology (Bauer and Dethlefsen, 1981), because total RNA content is stained by AO, and because there is less variation in quantum yield resulting from differences in base composition or conformation of RNAs than in the case of PY.

Differences in sensitivity of RNA detection. Because there is significant spectral overlap between AO bound to DNA and AO bound to RNA, the ability to detect small amounts of RNA under standard measuring conditions is better provided by the PY-Hoechst 33342 technique. Excitation of AO-stained cells at two different wavelengths, however, raises the sensitivity of the AO method to or above that of PY. Quantum yields and fluorescence intensities of cells stained with AO are higher than with PY.

Specificity in DNA content measurement. Hoechst 33342 is a more specific DNA stain than AO; the latter stains glycosaminoglycans (Darzynkiewicz and Kapuscinski, 1990) as well as DNA and RNA. Resolution of DNA measurements by AO in cells containing excessive amounts of glycosaminoglycans (primary fibroblasts, mast cells, and keratinocytes) is therefore low.

Analysis of RNA conformation. Both AO and PY can be used to reveal changes in the conformation of RNA. The PY-Hoechst 33342 technique detects changes associated with assemblage of polyribosomes (Traganos et al., 1988), whereas the AO method can be applied to measure the degree of double strandedness of RNA and its sensitivity to heat denaturation (melting profile).

Instrument requirements. The AO methodology (Basic Protocol 1, Alternate Protocol) has the advantage of requiring simpler and less costly instruments. The protocol can be performed with excitation at a single wavelength or with a mercury lamp instead of a laser.

RNA content standards

The lymphocytes may also serve as a calibrator of DNA content, to estimate the DNA index from the mean (modal) intensity of the green fluorescence of the G₀/G₁ population.

Applications of RNA analysis

Differences in RNA content between individual cells have two different origins. One is associated with the tissue type- or cell differentiation-related constitutive level of the translational activity. Thus, cells known to secrete, or produce for internal use, large quantities of tissue-specific protein (e.g., plasma cells or neurons) are generally characterized by high RNA content. In these cases the cellular RNA content is a reflection of a phenotype of the differentiated cell. Its measurement, therefore, can discriminate between cells of different tissues in the sample or be a marker of cell differentiation. The most common application of RNA measurement, in this context, is to identify reticulocytes, the immature red blood cells which retain RNA; in contrast, mature red blood cells have no measurable RNA (Tanke et al., 1980).

The second cause of variability in RNA content is related to cell reproduction. Dividing cells double their constituents, including the number of ribosomes, during the cell cycle. Progression through the cell cycle is thus associated with an increase in cellular RNA content, which occurs throughout interphase at a relatively constant rate proportional to the rate of cell proliferation (Darzynkiewicz et al., 1984a). Cellular RNA content, therefore, is a reflection of cell maturity in the cycle, and as such allows the discrimination of early-G₁ from late-G₁ cells (Darzynkiewicz et al., 1980). Because growth in cell size (number of ribosomes) and the rate of proliferation are generally coupled, the RNA parameter, therefore, is indirectly a marker of cell proliferation.

Noncycling cells withdrawn from the cell cycle (quiescent cells, G₀, G_1Q) have on average 5- to 10-fold fewer ribosomes than their cycling counterparts (Johnson et al., 1974). Thus, the difference in RNA content allows one to identify the noncycling cells and can be used as a marker of their mitogenic stimulation (Darzynkiewicz et al., 1976). Tumorous transformed cells, on the other hand, even noncycling ones, are characterized by an RNA content significantly higher than that of normal quiescent cells (Stanners et al., 1979). There is a growing body of evidence that the RNA content of tumor cells has prognostic value in many malignancies (reviewed by Darzynkiewicz, 1988).

Critical Parameters and Troubleshooting

1. Preservation of intact cells during permeabilization with Triton X-100. Disintegration (lysis) of unfixed cells during permeabilization by Triton X-100 is prevented by the presence of serum or serum albumin (Darzynkiewicz et al., 1976). It is recommended, therefore, to have the cells suspended in PBS or culture medium that contains 10% to 20% (v/v) serum or 1% (w/v) bovine serum albumin prior to addition of the permeabilizing solution. Furthermore, vigorous shaking, pipeting, or vortexing cell suspensions after addition of the detergent should be avoided (Darzynkiewicz et al., 1976).

2. Critical concentration of AO. Differential staining of RNA versus DNA requires a proper concentration (~20 mM) of free (unbound) AO in the final staining solution as well as at the time of fluorescence measurement, i.e., at the moment of cell intersection with the laser beam in the flow cytometer. The following problems associated with this requirement may occur:

(a) When the cell number (density) in the original suspension exceeds 2×10⁶ cells/ml (or even less when cells are highly hyperdiploid and/or have excessive RNA content), the amount of bound AO is high and therefore the free dye concentration may be significantly reduced (the "mass action" law). RNA denaturation is then incomplete, and some RNA can stain green. In such cases, dilute the cell suspension to obtain a lower concentration.

(b) With some instruments (e.g., most cell sorters) in which cell measurements take place in air outside the nozzle, a significant diffusion of dye from the sample to the sheath fluid takes place before the cell reaches the laser beam (see Fig. 1.1.6). This breaks the equilibrium and lowers the actual AO concentration in the sample at the time of cell measurement. Dye diffusion is also a problem in some instruments that have narrow sample streams and long flow channels (e.g., Cytofluorograf 50 made by Ortho Diagnostics). The solution is to increase the AO concentration in the AO solution (up to 20 mg/ml) and to increase the sample flow rate to compensate for the diffusion. Wherever possible, use channels with favorable geometry (wider sample stream and/or shorter distance between the nozzle and intersection with the laser beam). The optimal dye concentration for a particular instrument can be established by preparing a series of staining solutions with increasing AO concentrations (e.g., from 5 to 20 mg/ml) and determining the concentration at which cells in G₀/G₁ cell cluster have the same green fluorescence (the lowest coefficient of variation [CV] of the green fluorescence mean value, corresponding to a lack of correlation between green and red luminescence). On the bivariate DNA/RNA cytograms (see Figs. 7.3.1 and 7.3.2), the G₀/G₁ cell cluster ought to be horizontal (or vertical if axes are reversed) but never skewed (diagonal).

3. Overlap of red and green emission spectra of AO. One of the limitations of the AO technique is the relatively low sensitivity of RNA detection. This is primarily due to overlap of the emission spectra: the green fluorescence of AO intercalated to DNA has a long tail toward higher wavelengths. Therefore, RNA measurements in cells (or cell nuclei) characterized by a high DNA/RNA ratio lack sensitivity, being obscured by the high component of AO bound to DNA. There are two ways to improve the sensitivity of RNA measurements: (1) Use long-pass filters transmitting above 640 or 650 nm, rather than 610 or 620 nm, to measure red luminescence. This significantly reduces the DNA-associated spectral component. (2) When possible use two lasers for excitation, one tuned to 457 nm to excite the red luminescence and another tuned to 488 nm to excite the green fluorescence of AO.

4. Contamination of sample tubing by AO. Like other cationic, strongly fluorescing fluorochromes (e.g., rhodamine 123), AO is adsorbed to surfaces of the sample flow tubing. Its subsequent release from the tubing interferes with later sample measurement, especially with cells of low fluorescence intensity. To avoid this problem, after measuring samples stained with AO and prior to measuring samples stained with other fluorochromes, rinse the sample flow line with bleach (e.g., 10% Clorox), then 50% ethanol, then PBS, each for 10 min. Alternatively, if possible, have some sample flow tubing dedicated exclusively to AO use.

Differential staining with PY-Hoechst 33342

Because of these denaturing properties of PY, the RNA stainability is very sensitive to native RNA conformation. Under appropriate conditions, the procedure allows one to discriminate between polyribosomal RNA (which is more resistant to denaturation and shows more extensive double strandedness) and rRNA in dispersed ribosomes (Traganos et al., 1988). Thus the staining procedure can be used to measure disaggregation of polyribosomes occurring during mitosis or hyperthermia.

It should be stressed that PY taken up by live cells is partially localized in mitochondria and lysosomes (Darzynkiewicz et al., 1987). The specificity of staining of RNA with PY in live cells (Shapiro, 1981), therefore, is uncertain.

Anticipated Results

Stainability of RNA and DNA with PY and Hoechst 33342 is shown in Figure 7.3.2. The staining reaction is very sensitive to the conformation of RNA in the cell. Thus, as is evident, cells in mitosis (M) have lower stainability with PY than do G₂ cells, despite the fact that the RNA content of M cells is somewhat higher than that of most G₂ cells. The hypochromicity of RNA in M cells with PY is a consequence of the denaturing properties of the dye: RNA of polyribosomes (which are more numerous in interphase than in metaphase) is more resistant to denaturation and thus stains more intensively compared to RNA in mitotic cells. During mitosis, polyribosomes disaggregate and RNA of individual ribosomes is more extensively denatured by PY, leading to quenching of fluorescence (Traganos et al., 1988).

Time Considerations

Literature Cited

Crissman, H.A., Darzynkiewicz, Z., Tobey, R.A., and Steinkamp, J.A. 1985. Correlated measurements of DNA, RNA and protein in individual cells by flow cytometry. Science 228:1321-1324.

Darzynkiewicz, Z. 1988. Cellular RNA content, a feature correlated with cell kinetics and tumor prognosis. Leukemia 2:777-787.

Darzynkiewicz, Z. and Kapuscinski, J. 1990. Acridine orange: A versatile probe of nucleic acids and other cell constituents. In Flow Cytometry and Cell Sorting, 2nd ed. (M.R. Melamed, T. Lindmo, and M.L. Mendelsohn, eds.) pp. 291-314. Wiley-Liss, New York.

Darzynkiewicz, Z., Traganos, F., Sharpless, T., and Melamed, M.R. 1976. Lymphocyte stimulation: A rapid multiparameter analysis. Proc. Natl. Acad. Sci. U.S.A. 73:2881-2884.

Darzynkiewicz, Z., Sharpless, T., Staiano-Coico, L., and Melamed, M.R. 1980. Subcompartments of G₁ phase of the cell cycle identified by multiparameter flow cytometry. Proc. Natl. Acad. Sci. U.S.A. 77:6696-6699.

Darzynkiewicz, Z., Crissman, H.A., Traganos, F., and Steinkamp, J. 1984a. Cell heterogeneity during the cell cycle. J. Cell Physiol. 113:465-474.

Darzynkiewicz, Z., Traganos, F., Kapuscinski, J., Staiano-Coico, L., and Melamed, M.R. 1984b. Accessibility of DNA in situ to various fluorochromes: Relationship to chromatin changes during erythroid differentiation of Friend leukemia cells. Cytometry 5:355-363.

Darzynkiewicz, Z., Kapuscinski, J., Traganos, F., and Crissman, H.A. 1987. Application of pyronin Y (G) in cytochemistry of nucleic acids. Cytometry 8:138-145.

Johnson, L.F., Abelson, H.T., Green, H., and Penman, S. 1974. Changes in RNA in relation to growth of fibroblasts. I. Amounts of RNA in resting and growing cells. Cell 1:95-100.

Kapuscinski, J. and Darzynkiewicz, Z. 1987. Interactions of pyronin Y (G) with nucleic acids. Cytometry 8:129-137.

Kapuscinski, J. and Darzynkiewicz, Z. 1989. Structure destabilization and condensation of nucleic acids by intercalators. In Biological Structure, Dynamics, Interactions and Stereodynamics (R.H. Sarma and M.H. Sarma, eds.) pp. 267-281. Adenine Press, Schenectady, N.Y.

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Table 7.3.1 Spectral Characteristics of Dyes and Nucleic Acid-Dye Complexes
Dye or complex	Absorption maximum (nm)	Recommended excitation (nm)	Emission maximum (nm)	Recommended band-pass or long-pass filter (nm)
AO (monomer)	492	_	525	_
AO-ds DNA (intercalated)	502	488	520-524a	52015
AO-ss DNA (precipitated)	426-458b	457	630-644b	>640
AO-ss RNA (precipitated)	426-458b	457	630-644b	>640
PY	547	_	565	_
PY-ds DNA	547-563b	488-530	565-574b	>560
PY-ds RNA	547-563b	488-530	565-574b	>560
Hoechst 33342	355	_	467	_
Hoechst 33342-DNA	351	350-360	466	48015
aGreen fluorescence of AO-DNA has a long tail toward higher wavelengths. bValue depends on base composition.

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