Histoehemistry 46, 147--160 (1976) 9 by Springer-Verlag 1976

Further Observations on the Chemistry of Pararosaniline-Feulgen Staining J. E. Gill a n d M. M. Jotz Lawrence Livermore Laboratory, University of California Livermore, California 94550 Received September 3, 1975

Summary. Pararosaniline-Feulgen staining of cells in suspension produces nucleus- and chromatin-speeifie fluorescence as well as color. Experiments were designed to test postulated reaction mechanisms responsible for the fluorescent staining with the nonfluorescent pararosaniline. The reduction in fluorescent-staining intensity by pretreatment of cells with 2.2 x 10-2 M K2S205 tends to rule out the alkysulfonic acid pathway; conditions favoring the formation of this intermediate reduce staining intensity. The fluorescence enhancement, observed when cells stained in pararosaniline without K2S205 are post-treated with K2S205, suggests that there is an initial Schiff-base linkage between pararosaniline and an aldehyde of hydrolyzed ])NA, and that this linkage is stabilized in the presence of K2S~O5. Mierospeetrofluorometer measurements of cells stained at various pararosaniline concentrations in 2.2 • 10-2 M K2S205, show that the fluorescence emission maximum ranges from about 627 nm at 3.1 X 10-a M pararosaniline to about 604 nm at 3.1 x 10.5 M. All of the employed staining protocols appear to produce the same fluorescent product, perhaps a heterocyclie pyronin analog formed from pararosaniline. Flow microfluorometric analysis of cells stained in suspension verified that the relative fluorescence intensity represents relative DNA content. Staining at reduced pararosaniline concentration (3.1 X 10-~ M) reduces the coefficient of variation of the flow microfluorometric histograms, showing that maximum quantitation does not necessarily correlate with maximum staining intensity.

Introduction The p a r a r o s a n i l i n e - F c u l g e n r e a c t i o n for cellular D N A content, first described more t h a n 50 years ago (Feulgen a n d Rossenbeck, 1924), is still widely used today. This procedure gives reaction products t h a t fluoresce as well as absorb (Ploem, 1967), a n d so can be used with b o t h cytofluorometric a n d c y t o p h o t o m e t r i c instrumentation. M e a s u r e m e n t of relative D N A c o n t e n t s a m o n g a p o p u l a t i o n of cells stained with a p a r a r o s a n i l i n e - F e u l g e n procedure is employed i n both basic cell biology a n d clinical medicine. The relative D N A c o n t e n t of a cell identifies its position i n the cell cycle; thus p a r a r o s a n i l i n e - F e u l g e n stained cells can be analyzed for cell-cycle p a r a m e t e r s a n d p e r t u r b a t i o n s (Vendrely, 1971). Altered p a t t e r n s of D N A c o n t e n t a m o n g a p o p u l a t i o n of cells have b e e n associated with a v a r i e t y of malignancies, a n d m a y serve to i d e n t i f y such diseases as mycosis fungoides (Van Vloten et al., 1974). I g n o r a n c e of the p a r a r o s a n i l i n e - F e u l g e n reaction m e c h a n i s m a n d t h e structure of t h e reaction products has i m p e d e d more effective application of this procedure. There are two reaction schemes generally given credence (Pearse, 1968). These schemes share the decolorization of pararosaniline b y t h e addition of a sulfonic

148

J . E . Gill and M. M. Jotz

H2N~ - ~ / A 2 C ~ N H ~ - -

+ SO2 + H20

NH2 S03H H2N

NH2, + SO2

NH2

SO3H

H

I

I

0

II

+C-H i

R

NH2

/2--~ SO3H I jT--l~ HI H2N ~

~

OH ~ I

etc.

NH2

Fig. 1. Formation of an N-sulfonic acid-aldehyde. A molecule of SO2 first binds to the central carbon of pararosaniline, giving the colorless intermediate. A second molecule of SO2 then sulfonates one of the amino groups forming the Schiff reagent. This sulfonate group subsequently reacts with an aldehyde of hydrolyzed DNA, linking the pararosaniline to DNA (Pearse, 1968)

acid group to t h e central c a r b o n of pararosaniline. The schemes differ in t h e role p l a y e d b y SO 2 in linking t h e deeolorized p a r a r o s a n i l i n e to one or m o r e a l d e h y d e groups on t h e h y d r o l y z e d D N A . I n one scheme, a sulfonie acid g r o u p adds to a par~rosaniline a m i n o group, a n d s u b s e q u e n t l y binds to an a l d e h y d e , forming an N-sulfinie acid b r i d g e (Fig. 1). This r e a c t i o n is r e p e a t e d so t h a t t w o of t h e aniline groups are l i n k e d t h r o u g h such a b r i d g e to a l d e h y d e carbons of h y d r o l y z e d D N A . I n t h e o t h e r scheme, t h e r e is no a d d i t i o n of SOsI-I to t h e p a r a r o s a n i l i n e amino groups. I n s t e a d , excess SO 2 in t h e staining solution reacts with t h e a l d e h y d e s of t h e h y d r o l y z e d D N A to form an alkylsulfonie acid. The c a r b o n a d j a c e n t to t h e sulfonie acid t h e n links to t h e a m i n o n i t r o g e n of p a r a r o s a n i l i n e (Fig. 2). I n t h e first scheme, t h e Sehiff r e a g e n t is d o u b l y s u l f o n a t e d p a r a r o s a n i l i n e (N-sulfinie acid). I n t h e second scheme, t h e r e a g e n t consists of decolorized p a r a r o s a n i l i n e plus excess SO 2. W h i l e t h e r e is evidence f a v o r i n g b o t h m e c h a n i s m s (see Pearse, 1968 or Swift, 1955 for summaries), t h e r e are o b s e r v a t i o n s t h a t n e i t h e r r e a c t i o n scheme can

The Chemistry of Pararosaniline-Feulgen Staining

II0 C-HI +

149

OH I H2S03

~ H-~-S03H

R

R

OH 1 H-#-SO3H+ H2N~;~ R

~-NH2

NH2

NH2

Fig. 2. Formation of an alkylsulfonie acid. A molecule of SO2 in aqueous solution reacts with the aldehyde carbon of hydrolyzed DNA to give an alkylsulfonie acid. Next the sulfonated carbon joins with an amino group of deeolorized pararosaniline, and the latter then reverts to the colored form (Pearse, 1968) explain. Barka and Ornstein (1960) found as many as six reaction products when the Sehiff reagent reacted with formaldehyde. Hiroaka (1973) has presented electrophoretie evidence that the Sehiff reagent itself contains four components, and there are at least five different reaction products between the Sehiff reagent and apurinie acid. Other problems continue to plague application of pararosaniline-Feulgen procedures. Staining intensities vary and eontribu{e to standard errors or coefficients of variations of the order of 10% in cytophotometrie measurements (Bachmann, 1969). Optimization of the reaction has been on a trial-and-error basis without general eonsensus on the criteria for optimal staining. Cytoplasmic staining has been observed in clinical materials both in our laboratory and by Beyer-Boon (1974). I t must contribute to measured absorbanee or fluorescence values. The source of cytoplasmic staining is unknown. Literature on the chemistry of pararosaniline staining up to 1960 has been summarized by Kasten (1960). Since then there have been a number of papers (Jordanov, 1963; Sandritter et al., 1965; Deiteh et al., 1968; Andersson and Kjellstrand, 1971, 1972; Kiefer et al., 1972; Vahs, 1973) investigating hydrolysis conditions and recommending "long time" hydrolysis with 4 or 5 N HC1 at room temperature or slightly above. Absorption spectral studies of the reaction between sulfur dioxide, pararosaniline, and formaldehyde in solutions (Nauman et al., 1960) suggested that the mechanism for the reaction included the synthesis of a pararosaniline methylsulfonie acid. Itiroaka (1973) separated reaction products of pararosaniline, sodium bisulfate, and apurinie acid prepared under a variety of conditions, and concluded that the visible absorbing reaction products consist of pararosaniline molecules with two linkages to the apurinie

150

J.E. Gill and M. M. Jotz 0 [

SO3H

ii C-H R

NH2

H

so3H +

H2SO3

NH2

and/or

NH2

H

NH2

Fig. 3. Formation of a Sehiff-base linkage between decolorized pararosaniline and an aldehyde of hydrolyzed DNA. The data presented in Table 3 suggests this linkage may be subsequently stabilized in the presence of SOS, perhaps by sulfonation or hydrogenation

acid. The chemical form of the linkage was not made clear. Model system studies by van Duijn and co-workers have been used to determine the ratios of phosphate to pararosaniline in the reaction product (Hardonk and van Duijn, 1964), the optimal pH for rinsing the reaction product prior to dehydration, and the causes of variations in staining (Duijndam and van Duijn, 1973). Heinemann (1970) has published data on the reaction between "degassed" Schiff reagent and formalin, which he feels supports the conjecture that an alkylsulfonic acid is a precursor to the final reaction product. The spectral data could also be interpreted as evidence for a slow reaction linking decolorized pararosaniline and formalin without sulfonation. Previous work from our laboratory (Gill and Jotz, 1974) has indicated that the N-sulfinic acid is not formed by SO s and pararosaniline under usual staining conditions, and that aldehydes will react with SO2 under such conditions, but slowly. These studies also revealed that the chemistry of the so-called fluorescent-"Feulgen" reaction with aeriflavine (Sprenger et al., 1971) is different from

The Chemistry of Pararosaniline-Feulgen Staining

151

t h e p a r a r o s a n i l i n e - F e u l g e n r e a c t i o n , in t h a t SO d is n o t r e q u i r e d for b r i g h t , specific s t a i n i n g of n u c l e i a n d c h r o m a t i n b y a c r i f l a v i n e . W e also n o t e d t h a t n e i t h e r p u r i f i e d p a r a r o s a n i l i n e n o r t h e Schiff r e a g e n t was f l u o r e s c e n t . T h e e x p e r i m e n t s p r e s e n t e d h e r e w e r e d e s i g n e d to t e s t w h i c h of t h e p r o p o s e d r e a c t i o n m e c h a n i s m s for p a r a r o s a n i l i n e - F e u l g e n s t a i n i n g c a n a c c o u n t for t h e f l u o r e s c e n t r e a c t i o n p r o d u c t . O u r d a t a e x c l u d e s b o t h of t h e u s u a l l y c i t e d r e a c t i o n mechanisms. The reaction pathway most consistent with our data involves the f o l l o w i n g s t e p s : (1) A c o v a l e n t l i n k a g e is f o r m e d b e t w e e n p a r a r o s a n i l i n e a n d a n a l d e h y d e of h y d r o l y z e d D N A , p e r h a p s t h r o u g h t h e f o r m a t i o n of a Schiff base, as s h o w n in Fig. 3. (2) T h i s l i n k a g e is s t a b i l i z e d in t h e p r e s e n c e of SO 2. (3) T h e b o u n d p a r a r o s a n i l i n e is c o n v e r t e d i n t o a f l u o r e s c e n t c o m p o u n d .

Materials and Methods Preparation o/ Pararosaniline (C. I. 42,500) Staining Solutions. Schiff's reagent was prepared according to Graumann (1953). 0.1 gram of basic fuchsin (Allied Chem. Corp. ,Morristown, N. j.)l was dissolved in 15 ml of 1 N HC1, then combined with 85 ml of 2.6 X 10-2 M K2S20~ in distilled water, and stored overnight in the dark. The solution was shaken with 1 g of activated charcoal for two minutes, then filtered through 0.5-~z-Solvinert filters (Millipore Corp., Bedford, Mass.). Parasosaniline solutions without SO 2 were prepared in the same way, except that K2S205 was omitted. These solutions had impurity fluorescence intensities less than Raman scatter at all excitation wavelengths from 350 600 nm. Standard Staining Protocol. The standard protocol is a modification of the protocol of B5hm and Sprenger (1968), and follows the flow chart shown in Fig. 4. Spectroscopy. Absorption spectra of dyes and stains were obtained with a Beckman Acta V spectrophotometer. The concentration of pararosaniline was determined spectrophotometrically based on the molar extinction coefficient of 6.6 x 104 at 538 nm (Perkampus et al., 1971), Fluorescence analyses of dyes and stains were made with an Aminco-Bowman spectrophotofluorometer. Microscopy. Fluorescence, phase, and bright-field microscopy were done with a Zeiss Universal equipped for epi-excitation of fluorescence. Transmitted phase contrast or brightfield illumination was used to locate and identify ceils. Simultaneous epi-illumination for fluorescence and transmitted illumination were used to identify fluorescent-stained nuclei and chromatin within cells, and to verify that cytoplasm was present but unstained. The light source for fluorescence illumination was a 200-W-mercury lamp powered by a D.C. supply. The exciter filters were a K P 600 and a K P 546; the dichroic mirror in the epi-illuminator reflected wavelengths shorter than 580 nm, and transmitted longer wavelengths; the barrier filter was a Zeiss # 58. The light source for phase and bright-field illumination was a 60-W tungsten lamp. The intensity of transmitted illumination could be continuously adjusted by rotating one polarizing filter with respect to another. Mierospectrofluorometry. Fluorescence emission spectra from individual cells were obtained from a Princeton Applied l~esearch optical multichannel analyzer-spectrometer coupled to a Zeiss Axiomat, with a mechanical and optical interface. The optical multichannel analyzer consists of a linear array of photoelectric detectors, each of which independently measures light intensity as a function of time. Coupling such an analyzer to a spectrometer (wavelenghtdispersive device) provides two advantages for microspectrofluorometry, where intensity is measured as a function of wavelength: (1) detection and display of an entire fluorescence spectrum simultaneously in real time, and (2) elimination of spectral distortion as a result of sample fading as its fluorescence is scanned. The spectrometer analyzer contains 500 channels with a wavelength dispersion of 0.49 nm per channel. New spectra are read out every 33 ms and may be displayed in real time or stored in memory. A second memory bank permits back1 t~eference to a company or product name does not imply approval or recommendation of the product by the University of California or the U.S. Energy Research & Development Administration to the exclusion of others that may be suitable.

152

J.E.

Gill a n d M. M. J o t z Pool f i x e d c e l l s

Divide sample into N s i l i c o n i z e d glass tubes Clinical

centrifuge,

3000 R.P.M., 5 min.: 20~

Decant supfrnatant

Resuspend cells in 10 ml, Ca Mg:ree phosphate buffered saline Hold, 5 min., 20~ Centrifuge, 3000 R.P.M., 5 min., 20~ Decant sup;rnatant Resuspend cells in 0.2 ml 0.05 N He1 To -N tubes add ,5 ml 4 N HCl Hold, 100 min., 28~ Centrifuge, 3000 R.P.M., 5 min., 20~ Oecant supfrnatant Resuspend cells in 0.2 ml 0.05 N HCl To N tubes, add 5 nil 3.1 • ]0 -3 N pararosaniline, 2.2 • 10-2 M K25205 Held, 50 min., 20~

in the dark

Centrifuge, 3000 R.P.M., 5 min., 20~ Decant supfrnatant Resuspend cells in 5 ml acid alcohol

Held, 5 min., 20~

in the dark

T

Centrifuge, 3000 R.P.M., 5 min., 20~ Decant sup~rnatant Resuspend cells in 5 ml acid alcohol Hold, 5 min., 20~

in the dark

Centrifuge, 3000 R.P.M., 5 min., 20~ Decant

supfrnatant

Resuspend in 4 ml d i s t i l l e d

water f o r analysis

Fig. 4. A flow c h a r t g i v i n g t h e s t a n d a r d p a r a r o s a n i l i n e - s t a i n i n g protocol, a d a p t e d f r o m B 6 h m a n d S p r e n g e r (1968)

g r o u n d s u b t r a c t i o n . T h e microscope p r o v i d e s c p i - i l l u m i n a t i o n for fluorescence to m a x i m i z e s i g n M / b a c k g r o u n d , a n d t r a n s m i t t e d i l l u m i n a t i o n for selection a n d p o s i t i o n i n g of samples. T h e p o r t i o n of t h e i m a g e i n p u t t e d to t h e s p e c t r o m e t e r is d e t e r m i n e d b y a p e r t u r e s of k n o w n size a n d position. I n addition, t h e field of view c a n be p h o t o g r a p h e d . T h e m e c h a n i c a l i n t e r f a c i n g acts as a s u p p o r t , w i t h m i c r o m e t e r - c o n t r o l l e d p o s i t i o n i n g for t h e s p e c t r o m e t e r . T h e optical i n t e r f a c i n g is a lens t h a t d e m a g n i f i e s t h e e x i t p u p i l of t h e final i m a g e - f o r m i n g lens in t h e microscope a n d focuses t h i s i m a g e o n t h e e n t r a n c e slit of t h e s p e c t r o m e t e r . E m i s s i o n s p e c t r a were corrected for i n s t r u m e n t response o n t h e basic of a c a l i b r a t e d t u n g s t e n l a m p e m i s s i o n s p e c t r u m (Parker a n d R e e s , 1960). Flow Micro/luorometry. T h e F M F a n a l y s e s were p e r f o r m e d on a m a c h i n e (Van Dilla et al., 1973) a t L a w r e n c e L i v e r m o r e L a b o r a t o r y of design s i m i l a r to t h e F M F I I d e v e l o p e d ( H o l m a n d C r a m , 1973) a t t h e Los A l a m o s Scientific L a b o r a t o r y . T h e correlation b e t w e e n F M F m e a s u r e m e n t s of fluorescence i n t e n s i t y a n d relative D N A c o n t e n t s of F e u l g e n - s t a i n e d cells h a s b e e n d i s c u s s e d ( K r a e m e r et al., 1972). F l u o r e s c e n c e w a s e x c i t e d w i t h t h e 5 1 4 . 5 - n m line f r o m a n

The Chemistry of Pararosaniline-Feulgen Staining

153

argon ion laser, and observed through a Corning glass filter, C.S. 3-67, with an RCA C71461~ photomultiplier tube. Cells. Chinese hamster ovary (line CHO) cells (Puck et al., 1958) were grown routinely in suspension culture, in ~-minimal essential medium (Stanners et al., 1971) supplemented with antibiotics and 10% fetal calf serum (Flow Laboratories, Rockville, Md.).

Results The CHO cells grown in suspension culture were stained with pararosaniline i n suspension to see whether fluorescent staining would occur a n d w h e t h e r SO 2 was required for fluorescence. Cells t a k e n directly from t h e staining solution with K2S205 a n d e x a m i n e d u n d e r t h e microscope showed nucleus- a n d chromatinspecific bright red fluorescence a n d nucleus- a n d chromatin-specific purple coloring. Cells t a k e n directly from t h e staining solution w i t h o u t K2S~O 5 a n d e x a m i n e d u n d e r t h e microscope showed nucleus- a n d chromatin-specific weak red fluorescence a n d no color. Cells carried t h r o u g h the complete s t a n d a r d - s t a i n i n g protocol appeared as described above. Cells exposed to pararosaniline with a n d w i t h o u t K2S205 were e x a m i n e d with a microspectrofluorometer to see w h e t h e r the different staining protocols gave different reaction products with different emission m a x i m a . F i g u r e 5 shows t h a t pararosaniline with K2S20 5 generates a single fluorescence m a x i m u m at a p p r o x i m a t e l y 627 n m . P a r a r o s a n i l i n e w i t h o u t K2S205 generates a single fluorescence m a x i m u m at a p p r o x i m a t e l y 595 nm. F M F m e a s u r e m e n t s showed t h a t omission of K 2$205 from the staining solution reduced the fluorescence i n t e n s i t y to 5% of t h e controls. T h e d a t a on relative brightness a n d spectra are s u m m a r i z e d in T a b l e 1. Table 1. K2S205 dependence of fluorescence from p~rarosaniline-stained CHO cells 30 minute pretreatment

Staining Microscopic appearance concentrationa of nuclei and chromatinb

Microspectrofluorometer emission maximum (nm)

FMF relative brightness

K2S205 (M)

Color

Fluorescence

None

2.2 • 10-2

Pale purple color

Bright red

624 628

100

None

0

No color

Red

592 598

5

2.2 • 10-2 M K2S205 n O.05 N HCl

2.2 • 10-2

Pale purple color

Bright red

618 621

69

2.2 • 10 2 M K2S205 in 0.05 N HC1

0

No color

Weak red

592-598

4

0.05 N HC1

2.2 x 10-2

Bright red

623-624

106

0.05 N HC1

0

Weak red

592 598

7

Pale purple color No color

a Pararosaniline concentration was 3.1 • 10 3 M before charcoal treatment. b Cytoplasm was free of color and fluorescence.

154

J . E . Gill a n d M. M. J o t z Wavelength - 526 lO

550

nm

600

650

700

l

i

[

770

\

cr

\

c

/

o

\

/

W-

>~

0 1.

i

\

I

i

I

1.7 Wavenumber

1.5 --

i .3

~m- l

Fig. 5. Corrected fluorescence emission spectra from single pararosaniline-stained CttO cells: (--) fluorescence from a cell stained in 3.1 • 10 -3 M pararosaniline w i t h 2.2 • 10 2 M K2S205, a n d (---) fluorescence from a cell stained in 3.1 • 10 -a M pararosanilinc w i t h o u t K2S205

To test the role of acid hydrolysis on the specificity, intensity, and chemistry of the staining, separate aliquots of cells were either hydrolyzed in 4 N HC1 or held in 0.05 N HC1, then stained in pararosaniline with or without KeS205. Microscopic examination revealed that unhydrolyzed cells stained in pararosaniline with K2S205 had weak red fluorescence throughout and no visible color. Unhydrolyzed cells stained in pararosaniline without K2S~O 5 had very weak red fluorescence throughout and no color. Control aliquots of cells, subjected to hydrolysis followed by staining in pararosaniline with KeS205 gave cells with nucleus- and chromatin-specific bright red fluorescence and nucleus- and chromatinspecific purple coloring. There was no detectable cytoplasmic fluorescence or color. Hydrolyzed cells stained in pararosaniline without K2S~O 5 had nucleus- and chromatin-specific weak red fluorescence and no color. The FMF measurements showed t h a t omission of hydrolysis reduced the relative brightness to 7 % of the controls, omission of K 2S 205 from the staining solution reduced the relative brightness to 6 % of

The Chemistry of Pararosaniline-Feulgen Staining

155

Table 2. Staining concentration dependence of color and fluorescence Staining concentration a

Microscopic appearance of nuclei and chromatinb

Pararosaniline (M)

K2S2Q

Color

Fluorescence

3.1 • 10-a

2.2 X 10 2

Purple

Bright red

0.93 X 10-a

2.2 • 10-2

Purple

3.1 • 10-4

2.2 • 10-2

Pale Purple

0.93 • 10-~

2.2 • 10-2

:No color

Bright redorange

Microspectrofluorometcr emission maximum (nm)

(M)

FMF analysis Relative Coefficient brightof variation hess (%)

624 628

100

5.4

Bright red

617-618

100

5.1

Bright red

615 617

80

4.1

610

39

5.1

17

3.1 • 10-5

2.2 • 10-2

No color

Red-orange

604

0

2.2 • 10-2

No color

No fluorescence

Not measurable

0.93 X 10-a

0.66 • 10-2

Pale purple

Bright redorange

614

87

4.9

3.1 x 10 4

2.2 • 10-3

No color

Red-orange

604-606

23

15.0

0.93 X 10-4

0.66 • 10-a

No color

Very faint red Not measurable

2

13.1

3.1 X10 5

2.2 X10 -4

No color

:No fluorescence

0.6

24.0

Not measurable

0

7.6 :Not measurable

a Before charcoal treatment. b Cytoplasm was free of color and fluorescence.

t h e controls; omission of b o t h r e d u c e d t h e r e l a t i v e brightness to 1% (or less) of t h e controls. T h e fluorescence emission m a x i m u m of h y d r o l y z e d cells was at a b o u t 627 or 595 n m d ep en d i n g on w h e t h e r or n o t t h e r e was K2S205 in t h e staining solution. T h e fluorescence emission m a x i m a of t h e u n h y d r o l y z e d cells was at a b o u t 595 nm, regardless of t h e presence or absence of K2S205 in t h e staining solution. To test w h e t h e r p r e t r e a t m e n t of h y d r o l y z e d cells with K2S205 in 0.05 N HC1 would p r o m o t e t h e s u b s e q u e n t staining with pararosaniline, cells were p r e t r e a t e d with 2.2 • 10 -2 M K2S~O 5 in 0.05 N HC1, or w i t h 0.05 N HC1 only, an d st ai n ed in pararosaniline with or w i t h o u t K2S205. Control samples were stained with no p r e t r e a t m e n t . P r e t r e a t m e n t with 0.05 N HC1 slightly increased t h e r e l a t i v e brightness of cells stained in pararosaniline w i t h or w i t h o u t K2S205. I n contrast, pret r e a t m e n t with K2S205 in 0.05 N HC1 r e d u c e d t h e r e l a t i v e brightness of cells stained in pararosaniliae, with or w i t h o u t K2S205. W i t h e i t h e r p r e t r e a t m e n t , omission of K2S~O 5 f r o m t h e staining solution r e d u c e d t h e r e l a t i v e brightness b y a b o u t 95%. N e i t h e r of t h e p r e t r e a t m e n t s h a d a n y significant influence on t h e fluorescence emission spectrum. This d a t a is also s u m m a r i z e d in Table 1. To test w h e t h e r t h e fluorescence emission m a x i m u m was influenced by t h e staining concentration, different aliquots of CHO cells w e r e stained with t w o c o n c e n t r a t i o n series: 3.1 • 10 -5 M to 3.1 • 10 -s M pararosaniline in 2.2 • 10 -a

156

J . E . Gill and M. M. Jotz

Table 3. Effect of K2S20~ post-treatment on fluorescence of pararosaniline-stained CHO cells Staining 30 minute concentration~ posttreatment K2S205 (M)

Microscopic appearance of nuclei and chromatin

Microsl~ectrofluorometer emission maximum (nm)

FMF relative brighthess

Color

Fluorescence

2.2 • 10 2

2.2 x 10 ~ M KeS205 in O.O5 N HC1

Purple

Bright red

624 628

103

2.2 • 10-z

0.05 N HC1

Purple

Bright red

627-628

97

2.2 • 10 2 0

None

Purple

Bright red

624 628

100

2.2 • 10 2 M K2S205 in O.O5 N HC1

No color

Red

604 605

17

0

0.05 N HC1

No color

Weak red

592-598

4

0

None

No color

We~k red

592 598

4

a Pararos~niline concentration was 3.1 x 10-3 M before charcoal treatment. b Cytoplasm was free of color and fluorescence.

M K2S205, a n d 3 . 1 • 5M to 3 . 1 • - 3 M pararosaniline in 2 . 2 • - 4 M to 2 . 2 • 10-e M K2S20 5. Staining conditions were as described in Materials a n d Methods. R e d u c i n g the pararosaniline c o n c e n t r a t i o n with c o n s t a n t K2S~O 5 does shift the fluorescence emission m a x i m u m from 627 n m to 604 n m , a n d reduces the color a n d relative brightness. E l i m i n a t i n g the pararosaniline from t h e staining solution eliminates both color a n d fluorescence, l~educing both t h e pararosaniline a n d the K2SeO 5 concentrations reduces the color a n d the fluorescence i n t e n s i t y a n d shifts the s p e c t r u m m u c h more a b r u p t l y ; at t h e lowest concentration, no fluorescence can be detected. The F M F histograms of cell count versus fluorescence i n t e n s i t y were a n a l y z e d for coefficient of val-iation of the G-1 peak, to see whether the reduced a b s o r p t i o n b y the cells would reduce the spread in relative brightness. These data, along with relative brightness, emission m a x i m a , a n d appearance u n d e r the microscope, are given i n Table 2. To test w h e t h e r SO 2 m i g h t serve to stabilize the b i n d i n g of pararosaniline to hydrolyzed c h r o m a t i n and/or to react with b o u n d pararosaniline to promote color a n d fluorescence, cells s t a i n e d i n pararosaniline with or w i t h o u t K2S20 ~ were rinsed for 10 rain i n 0.05 N HC1, t h e n p o s t - t r e a t e d with 2.2 • l 0 -2 M K2S~O 5 in 0.05 N HC1, or with 0.05 N HC1 only. The relative brightness of t h e fluorescence from cells s t a i n e d in p a r a s o n a l i n i n e with K2S205 was increased b y 3% b y the K2S205 p o s t - t r e a t m e n t a n d decreased 3 % b y the 0.05 N HC1 p o s t - t r e a t m e n t . The relative brightness of fluorescence from cells s t a i n e d in pararosaniline w i t h o u t K2S20 ~ was increased 400% b y t h e K2S20 5 p o s t - t r e a t m e n t a n d u n c h a n g e d b y the 0.05 N HC1 p o s t - t r e a t m e n t . The fluorescence emission s p e c t r u m of cells stained in pararosaniline with K2S20 ~ was n o t altered b y either p o s t - t r e a t m e n t . Also, the fluorescence emission s p e c t r u m of cells s t a i n e d i n pararosaniline w i t h o u t K2S~O 5 was n o t altered b y the 0.05 N HC1 p o s t - t r e a t m e n t . However, the fluores-

The Chemistry of Pararosaniline-Feulgen StMning

157

cence emission of cells stMned in pararosaniline without KeS205 was shifted from about 595 nm to 604--605 nm by the K~S205 post-treatment. These data are summarized in Table 3. Discussion Of the several mechanisms proposed for the chemistry of pararosaniline staining, the two most accepted are: (1) the formation of N-sulfinic acid-aldehydes and (2) the formation of alkylsulfonic acid (Figs. 1-2). Another reaction t h a t appears possible is (3) the linking of a pararosaniline amino group to the aldehyde carbon, forming a Schiff base, followed by the conversion of pararosaniline to a heterocyclic fluorescent compound (Figs. 3 and 6). Arguments for and against the first two mechanisms are well documented (Pearse, 1968). Arguments regarding the last mechanism will be presented in this discussion of our data. Pararosaniline-Feulgen staining of cells in suspension and subsequent analysis for fluorescence has not to our knowledge been reported previously. Our results show that the fluorescent product is produced during the staining itself and does not require subsequent exposure to acid alcohol or dehydration. PararosanilineFeulgen staining of cells in suspension provides nucleus- and chromatin-specific products for analysis of cellular DNA content: red fluorescence for flow microfluorometry as well as visible absorbance for microspectrophotometry. In a previous publication, we presented spectral evidence from solutions that sulfonation of the pararosaniline amino groups did not occur under the conditions of the stMning reaction (Gill and Jotz, 1974). By eliminating the first mechanism, we inferred the second to be the correct choice. However, the second mechanism predicts that pretreatment of DNA aldehydes with acidic SO~ should promote the binding of pararosaniline where SO 2 is otherwise deficient, and not alter the staining with the Sehiff reagent. The results presented in Table 1 contradict this prediction and indicate that pretreatment of hydrolysed cells with acidic SO 2 reduces fluorescent staining with pararosaniline. An alternative hypothesis, not yet tested, is that sulfonation of the aldehydes involves a transient intermediate product that promotes staining, and that this intermediate product changes within minutes to a product t h a t blocks staining. The different emission maxima seen from cells stained with or without K2S205 suggested that: (1) different products might be formed under different conditions or (2) one product is formed and its emission spectrum is concentration dependent. The data from cells stained with different concentrations supports the second hypothesis. There is no evidence to suggest more than one fluorescent product, however this possibility cannot yet be ruled out. The emission shift with high staining concentrations could be caused by: (1)reabsorption of fluorescence or (2) metachromasia of the fluorescent product. I n either case, the present best value for the emission m a x i m u m of the fluorescent product is 595:L3 nm, obtained at low staining concentrations. The concentration dependence experiment, summarized in Table 2 also shows t h a t for FMF analysis, staining with 3.1 • 10 -4 M pararosaniline in 2.2• 10 2 M K2S~O 5 gives a 24% smaller coefficient of variation than the conditions initially chosen by us, based on the work of B6hm and Sprenger (1968). Since the coefficient of variation represents the width of the G-1 peak in the DNA content histogram,

158

J.E. Gill and M. M. Jotz

/•NH2 NH2

~NH2

Fig. 6. Structures of substituted pararosaniline and a possible hetero-cyelic conversion product. Pararosaniline is nonplanar and nonfluorescent. The heterocyclic compound, where the central ring is completely closed, is a planar pyronin analog smaller values imply more accurate measurements. Thus the first three lines of Table 2 indicate that increased accuracy of measurement is correlated with reduced staining intensity. There is no reason to believe that fluorescent staining for DNA eonteIlt cannot be optimized still further. If the two widely proposed mechanisms cannot account for fluorescent staining, other mechanisms need to be considered. A third mechanism is the reaction of a pararosaniline amino group directly with an aldehyde of hydrolyzed DNA, forming a Sehiff base. This product would subsequently be stabilized in the presence of acidic SO2, perhaps by sulfonation or hydrogenation of the Schiffbase double bond, as shown in Fig. 3. This mechanism is supported by the observation that cells stained without K2S205, then post-treated with an acidic K2S205 solution, are 400% more fluorescent than cells stained without K2S205, then post-treated with 0.05 N HC1 only. Since all the stained cells were rinsed in 0.05 N HC1 prior to the post-treatment, it is unlikely that the enhanced fluorescent staining could be ascribed to the reaction of unbound pararosaniline with sulfohated DNA. Further, it appears that the fluorescent product formed upon acidic SO 2 post-treatment of cells stained without K2S205 is the same as those formed in the standard reaction protocol. The post-treated cells have about 20 % of the brightness of cells stained with the standard protocol, and emit at 605 nm compared with 627 nm for cells stained with the standard protocol. Thus the fluoreseenee characteristics of these post-treated cells are the same as the fluorescence characteristics of cells stained at reduced pararosaniline concentrations, with K2S2Oa: Table 2, lines 5 and 8. The experiments with unhydrolyzed cells shows that hydrolysis not only promotes fluorescent and absorption staining of ehromatin, but eliminates cytoplasmic fluorescent staining. The two results that stand out most clearly are: (1) that the reaction meehanism for the fluorescent product is different from the N-sulfinic acid-aldehyde intermediate or the alkylsulfonic acid intermediate pathways. (2) The identity of the fluorescent product is unknown. While the Sehiff-base stabilization mechanism would explain the binding on the basis of our data, it would not of itself explain the generation of a fluorescent reaction product. I t m a y be that within the cells, pararosaniline undergoes a ring closure analogous to the conversion of Malachite Green to Rosamine. This would give the planar heteroeyclie structure needed for the fluorescence (F6rster, 1951), and would also suggest that the fluorescent

The Chemistry of Pararosaniline-Feulgen Staining

159

product is different from the a b s o r p t i o n product. The data needed to resolve these questions will be o b t a i n e d b y comparing excitation a n d absorption spectra of pararosaniline stained cells, a n d b y comparing e x c i t a t i o n a n d emission spectra of pararosaniline s t a i n e d cells with excitation a n d emission spectra of p o s t u l a t e d reaction products. Acknowledgements. We thank P. Lindl and L. Thompson for supplying CHO cells in spinner culture, L. Steinmetz and B. Wallin for operating the flow microfluorometer, and D. Davis and W. Jackson for help in assembling the microspectrofluorometer. This work was performed under the auspices of the U.S. Energy Research and Development Administration and with the support of USPHS Grant 1R01 GM20901.

References Andersson, G. K. A., Kjellstrand, P. T. T. : Exposure and removal of stainable groups during Feulgen acid hydrolysis of fixed chromatin at different temperatures. Histoehemie 27, 165-172 (1971) Andersson, G. K. A., Kjellstrand, P. T. T. : Influence of acid concentration and temperature on fixed chromatin during Feulgen hydrolysis. Histochemie 30, 108-114 (1972) Bachmann, K. : On the interspecific comparison of nuclear DNA amounts using the Feulgen and gallocyanin chromalum methods./-listochemie 17, 145-150 (1969) Barka, T., Ornstein, L. : Some observations on the reaction of Schiff reagent with aldehydes. J. Histochem. Cytochem. 8, 208--213 (1960) Beyer-Boon, T., Ploem, J. S. : A new type of staining for quantitative microfluorometric studies of cytology specimens. The Fifth International Congress of Cytology. Abstract 10. Miami Beach, Florida 1974 BShm, N., Sprenger, E. : Fluorescence cytophotometry: A valuable method for the quantitative determination of nuclear Feulgen-DNA. Histochemie 16, 100-118 (1968) Deitch, A. D., Wagner, D., Richart, R. M. : Conditions influencingthe intensity of the Feulgen reaction. J. Histochem. Cytochem. 16, 371-379 (1968) Duijndam, W. A. L., van Duijn, P. : The dependence of the absorbance of the final chromophore formed in the Feulgen-Schiff reaction on the pit of the medium, ttistochemie 35, 373-375 (1973) Feulgen, R., Rossenbeck, It. : Mikroskopisch-chemischer Nachweis einer Nukleinsgure yon Typus der Thymonucleinsgure and die darauf beruhende elektive F~rbung von Zellkernen in mikroskopischen Pr~paraten. I-Ioppe-Seylers Z. physiol. Chem. 135, 203-248 (1924) F6rster, T. : Fluoreszenz organischer Verbindungen, p. 109 113. GSttingen: Vandenhoeck and Ruprecht 1951 Gill, J.E., Jotz, M.M.: Deoxyribonucleic acid cytochemistry for automated cytology. J. Histochem. Cytochem. 22, 470-477 (1974) Graumann, W. : Zur Standardisierung des Schiffschen Reagens. Z. wiss. Mikr. 61, 225-226 (1953) Hardonk, M. J., van Duijn, P. : A quantitative study of the Feulgen reaction with the aid of histochemical model systems. J. :Histochem. Cytochem. 12, 752-757 (1964) I-Ieinemann, R. L. : Comparative spectroscopy of basic fuchsin, Schiff reagent, and a formalinSchiff derivative in relation to dye structure and staining. Stain Technol. 45, 165-172 (1970) I-Iiroaka, T. : Feulgen nucleal reaction II. Approaches to the understanding of mechanism of in vitro reaction, ttistochemie 35, 283-296 (1973) :Holm, D. M., Cram, L. S. : An improved flow microfluorometer for rapid measurement of cell fluorescence. Exp. Cell Res. 80, 105-110 (1973) Jordanov, J. : On the transition of dcsoxyribonncleic acid to apurinic acid and the loss of the latter from tissues during Fculgen reaction I-]ydrolysis. Aeta histochem. (Jena) 15, 135-152 (1963) Kasten, F. It. : The chemistry of Schiff reagent. Int. Rev. Cytol. 10, 1-100 (1960) Kiefer, 1~., Kiefer, G., Sandritter, W. : Feulgenhydrolysekinetikin Eu- and Heterochromatin. Histochemie 80, 150-155 (1972)

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Kraemer, P. M., Deaven, L. L., Crissman, H. A., Van Dilla, M. A.: DNA constancy despite variability in chromosome number. In: Advances in cell and molecular biology (E. J. DuPraw, ed.), vol. 2, p. 47-108. New York: Academic Press 1972 Nauman, R.V., West, P . W . , Tron, F., Gaeke, G. C. : A spectrophotometric study of the Schiff reaction as applied to the quantitative determination of sulfur dioxide. Analyt. Chem. 32, 1307-1311 (1960) Parker, C. A., Rees, W. T. : Correction of fluorescence spectra and measurement of fluorescence quantum efficiency. Analyst 85, 587-600 (1960) Pearse, A. G. E.: Histochemistry, vol. 1. Boston: Little, Brown, and Co. 1968 Perkampus, H. ~I., Sandeman, I., Timmons, C. J. (eds.) : UV atlas of organic compounds. :New York: Plenum Press 1971 Ploem, J. S. : The use of a vertical illuminator with interchangable dichroie mirrors for fluorescence microscopy with incident light. Z. wiss. Mikr. 68, 129-142 (1967) Puck, T. T., Ciecura, S. J., Robinson, A. : Genetics of somatic mammalian cells. I I l . Long-term cultivation of euploid cells from human and animal subjects. J. exp. Med. 108, 945-955 (1958) Sandritter, W., Jobst, K., Rakow, L., Bosselmann, K. : Zur Kinetik der Feulgenreaktion bei verls Hydrolysezeit. Cytophotometrische Messungen im sichtbaren und ultravioletten Licht. Histochemie 4, 420-437 (1965) Sprenger, E., B6hm, :N., Sandritter, W. : Flow-through-cytophotometry for ultrarapid DNA measurement of large cell populations. Histochemie 26, 238-257 (1971) Stanners, C. P., Elieieri, G., Green, H. : Two types of ribosome in mouse-hamster hybrid cells. :Nature (Lond.) :New Biol. 230, 52-54 (1971) Swift, It. : Cytochemical techniques for nucleic acids. In: The nucleic acids (E. Chargaff and J. :N. Davidson, eds.), vol. II, p. 51-92. New York: Academic Press 1955 Vahs, W. : Die Bedeutung der Hydrolyse-Art in der Feulgen-Cytophotometrie yon Kernen mit unterschiedlichen Ploidiegraden. Histochemie ~3, 341-348 (1973) Van Dilla, M. A., Steinmetz, L. L., Davis, D. T., Calvert, R. :N., Gray, J. W. : High-speed cell analysis and sorting with flow systems: biological application and new approaches. I.E.E.E. Trans. Nue. Sci., :NS 21, 714-720 (1973) Vendrely, C. : Cytophotometry and histochemistry of the cell cycle. In: The cell cycle and cancer (R. Baserga, ed.), p. 227~68. New York: Marcel Dekker 1971 Vloten, W. A., van Duijn, P. van Schaberg, A. : Cytodiagnostic use of Feulgen-DNA measurements in cell imprints from the skin of patients with mycosis fungoides. Brit. J. Derm. 91, 365-371 (1974) Dr. J. E. Gill Department of Pathology University of Rochester School of Medicine and Dentistry Rochester, :New York 14642 USA Dr. M. M. Jotz Lawrence Livermore Laboratory University of California Livermore, California 94550 USA