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THE JOURNAl, Copyright oc H ISTOCHEMISTRY AND © 1977 b’ The Histochemical SIMULTANEOUS C YTOCHEMISTRY Society. STAINING ACIDS IN UNFIXED Sloan-Kettering Received for to chelating agents with cells is procedures May 10, that 1976, DNA metachromatic discussed measured in a flow employing acridine in terms of staining provided that acridine Center, is denatured RNA to ensure the T. SHARPLESS of deoxyribonucleic is described. Cells staining orange AND 25. No. 1. pp 46-56. 1977 Printed in USA. DEOXYRIBONUCLEIC ACRIDINE ORANGE SYSTEM’ Cancer publication fixed, but permeable, cells ionic detergent at low pH. differentially RIBONUCLEIC Z. DARZYNKIEWICZ, Memorial cells OF CELLS USING CYTOFLUOROMETRIC F. TRAGANOS, Simultaneous Vol. Inc. dye, New and M. D York, New in revised IN R. MELAMED York form A FLOW 10021 July 14, 1976 (DNA) and ribonucleic acid (RNA) in nonare made permeable by treatment with nonprior to, or during staining, by exposure of (native) and RNA (denatured) may be stained acridine orange. The fluorescence of individual cytofluorometer. A comparison between various staining orange or other intercalating dyes in unfixed cells specificity, cell permeability and preservation. Evidence staining of unfixed cells may be used as a simple, fast is is means of obtaining information on cell ploidy levels and cell cycle status from DNA measurements (green fluorescence), and cell transcriptional activity from RNA staining (red fluorescence), in human and murine cells lines, peripheral blood and bone marrow specimens from patients with leukemia and mitogenically (phytohemagglutinin) or antigenically (mixed lymphocyte culture) stimulated human peripheral blood cultures. Exposure of cells to detergent at low pH as an alternative to cell fixation or hypotonic treatment is proposed as a fast, convenient method of making cells permeable to dyes. Specific staining of’ nuclear acid (DNA)2 within individual deoxyribonucleic cells offers means level of their analysing distribution cell cycle. in Many dyes that Recently, introduced proteins fixed cells using DNA with stain ing of DNA correlate the position We and the various of cells and stages methods staining are of’ the ent employ the differentially dye propidium fluorescein for stained as Simultaneous ribonucleic acid (RNA) by which may cycling cells activity work was supported by U.S. having (G,, based on ble are cells. helical G2 use of single nucleic acids It compared. cells (G0) from The of’ acridine stain cycle of differ- transcriptional M). + cell dormant increased S, the differential a activities be useful to distinguish a minimal level of transcription he to its method is (AO) orange stranded as versus a dou- (2, 6, 7. 20). MATERIALS Public AND METHODS Cells: Human peripheral blood separated on ficoll-hypaque, were mal healthy donors. Lymphocytes DNA from differen- monocytes Health glutinin Service Grant 126CA14134 through the National Bladder Cancer Project. 2Abbreviations used in text: DNA, deoxyribonucleic acid; P1, propidium iodide; RNA, ribonucleic acid; AO, acridine orange, PHA, phytohemagglutinin; MLC, mixed lymphocyte culture; ALL, acute lymphocytic leukemia; F550, green fluorescence intensity measured in a band from 515-575 nm; F.,00, red fluorescence intensity measured in a band from 600-650 nm. Stit. (MLC) after 6 days (3). (ALL) tine 46 way from of patients before were and kindly or absence as previously mixed microtiter No attempt with acute in provided plastic. was prior made to Cells cul- remove prepared and in peripheral leukemia treatment Doctor to sampled lymphoblastic by (9). cultures were marrow VILO and described plates. bone after to of phytohemag- iradiation preparations. the cells. from norseparated lymphcoyte by in MLC from same 72 hr for cells mononuclear obtained were of adherence inactivated the monocytes blood for cells svere and lack presence (PHA) ilator ture, the by in the cultured This unfixed stain- and protein makes it possible metabolic state of the cell with in the cell cycle. propose a simple method individual also with (P1) isothiocyanate proteins. within transcriptional may in iodide stained The analysis of RNA in relation to the would be of special interest when cells intercalate into DNA (4, 5. 13. 15, 23). Steinkamp and Crissman (4, 22) a two-parameter analysis in which and counter ploidy of the DNA for the tially a with Z. Arlin. vincris- Cell STAINING IN CELLS USING designated CEM, derived from a childhood lymphoma progressing to ALL (12) and EL4, a murine T cell leukemia (19), were a gift of Doctor P. Ralph. Friend virus-induced murine erythroleukemia cells ACRIDINE 47 ORANGE were as follows: two-step, pH 3.0: Aliquots (0.2 ml, containing approximately 2-5 x 10 cells) were withdrawn from cultures and were added to 0.5 ml of a solution containing: 0.1% (v/v) Triton X-100 (Sigma Chemical Co., St. Louis, Mo.), 0.2 M sucrose, 10’ M EDTA and 2 x 102 M citrate-phosphate buffer, at pH 3.0 fluorescence is measured in two F530 (515-575 nm) and F>,00 (600650 nm) as the cells travel in single file fashion through the focused beam of an argon ion laser (488 nm), subtracted from background fluorescence, digitized and recorded for further analysis. Staining specificity with AO: Lymphocytes from PHA cultures (48 hr) were treated for 1 mm with a solution consisting of 0.1% (v/v) Triton X, 0.2 M sucrose and 20 mM citrate-phosphate buffer at pH 3.0. The cells were then centrifuged, suspended in 0.25 M sucrose-5 mM MgCl2-20 mM tris HC1 (pH 6.5) and incubated for 20 mm at 37#{176}Cin the absence of nucleases or with 10 units/ml of RNAse A (Worthing- (9). ton lines (10) were Doctor subcultured from a cell line maintained by C. Friend. Cell treatment and staining: The various staining protocols 1. AO: Triton dures X-100 at the was included indicated pH in the to increase various cell proce- permeability yet maintain cellular integrity (see Discussion). The chelating agent EDTA was used to facilitate RNA denaturation as previously described (7, 9). The cellswere stained one minute later by addition of 1 ml of a solution containing 0.002% (20 g/ml) AO (Polysciences Inc., Warrington, Pa.), 0.1 M NaCl and 102 M citrate-phosphate buffer, pH 3.8 (9, 11). Cations were included in the staining mixture to ensure staining specificity (see Discussion). The final AO concentration was approximately 4 x 10 -5M. IL AO: two-step, 0.1 N HC1: Aliquots of 0.2 ml of cells in media with 10% fetal calf serum were mixed with 0.3 ml of a solution containing: 0.1% (v/v) Triton X-100, 0.1 N HC1 and 0.15 N NaC1. After mixing and equilibration (about 15 sec), 1 ml of AO (5 g/ml) in 5 x 10 M EDTA, 0.15 N NaCl, 0.1 M phosphate-citrate buffer, pH 6.0 was added giving a final dye concentration of 1.1 x 10 M AO. III. AO: one-step: containing: 10cells 5 tg/ml M EDTA 2 in serum P1: for 5 mm resuspended in 0.05 after P1: two-step except that dye previously (Data multaneous cence individual low were cells Southboro, of green (1-19#{176}) forward in suspension. as diploid mean The sys- to that flow cytoSystems Inc., Mass.) minicom- provides and red light As population in cell decreased AO with red fluorescence By use specificity a in lished (9). the and by increased the red C was cells could to up (9). 16 hr a marked could be identified green (DNA) fluorescence in all the G-S of boundary in 0.2 C2 green tg/ml were the of stimulated increase of terminated M populatin -+- as a population level system hydroxyurea days cultures increase which as analysed 2 mM addition before hr synthe- by treatment no The 24 RNA in this at fluorescence, gluorescence) vinblastine pile estab- added delineated stimulated cultures with 24 hr of cultures. By three red ig/ml) transition, PHA after seen staining was fluorescence, population be agents, prevented G0 to C (RNA) of of the (9). (0.05 of cultures as dyes 87% cultures D the is a result while blocking stimulated Actinomycin therefore The of PHA initiation sis DNA to RNA variety and detergent as well fluorescence cellular is due of 88% with to nucleases 88% of the green interaction by treatment permeability and at least after Freehold, I (Worthington). F>,00 by 7%. Therefore, results at twice of the C0 (or C) the cells (9). RESUI.TS One-step and 1220 Corp., DNAse was model to a Nova the N NaCI, in principle (Bio/Physics N.J.) or with 4 x After centrifugation the cells were resuspended in an AO solution (staining method I) and the mean F,30 and F>,,0 for the diploid cell population was calculated. Cells treated with RNAse showed a minimal (3%) decrease in F,30 but an 87% decrease in F,,00 when compared to control cultures. In the presence of DNAse, the mean F,30 of units/ml N HC1 mg/ml of fluorescence: interfaced angle treated in 0.1 0.15 Biochemical 10 caused buffer. pH 6.0. (6). A research General, contain- citrate Cells is identical measurements and solution cenwere the bands, (increased were pellets of 0.05 buffer, FC2000 N.Y.), M). in 5 x 10- 3M EDTA, study X-100, ( 18), wavelength (9). added to 0.2 ml of 10% fetal calf procedure a concentration described fluorometer Mahopac. puter Triton sodium N HC1: measurement in this solution (13). staining M phosphate-citrate Analysis, the 0.1 AO for AO used AO (v/v) iodide in a 0.1% of Krishan P1 at substituted tem an 1.2 x 10 suspensions 150 g and the cell at two-step, in the 0.1 0.07% a propidium mg/ml the method V. of AO concentration one-step: Cell (final trifuged AO, ml and 0.15 N NaC1 was with approximately media IV. ing 0.6 described si- scatter previously B shows staining wide for AO a comparison procedure use using employing and a technique a DNA stain of from a patient with ALL blood suspended phate buffered saline and 10% AO solution detergent cells human mixed peripheral with an (staining permeable method to the dye III) in (13). A leukocytes in fetal and 1A a single-step AO and P1 as Figure P1: between sample was fluores- staining: calf phosserum containing to to make a chelating the 48 TRAGANOS ET AL. cells while with little higher . red cycling CV 7 3 B compared IL C 0 (I, a, U be CV 8.3 using due lack to lysis 1. Computer and RNA drawn distribution shift cells (AU.) histogram of peripheral of the blood DNA not leukocytes and Methods). B, DNA distribution of cells stained with P1 in 0.1% citrate (13) (staining method DNA distribution of cells treated with 0.1N HC1 prior to staining with 0.05 mg/ml P1 in 5 x 10’ M EDTA, 0.15 N NaC1, 0.1 M phosphate-citrate buffer, pH 6.0 (staining method V). The coefficient of variation (CV) for the G, population are indicated. IV). C, agent (EDTA) which preferentially hypotonic microscopic (Table IV) after (Fig. of’ AO 6 hr (Fig. This A stained in the shift stained conditions (Fig. examination more 2). of culture 2A). preparation AO: staining: and red of three (staining method conditions which be was with P1 2B). Fluoresof specimens I). Two-step hypotonic (staining cells could both ways revealed that the detergent cells remained intact, while cells in a hypotonic medium were extremely the vast majority of chromosomes apfree or in small clumps on the slide (Figure 3A-C) from a patient gent solution solution and pro- IV) to deterabove might of mitotic distribution in the any double-stranded RNA (7, 9) (Fig. 1A). Staining was performed at pH 4.0. Parallel samples were stained with P1 in the presence of citrate the staining III and noted hypotonic observed observed treated treated stained fragile, pearing one-step loss of colcemid scatterplots histograms denatures two methods differences and in was under cence from a patient with ALL. A, green (DNA,--) and red (RNA, ) fluorescence distributions of cells stained by the one-step AO procedure at pH 4.0 (see Materials suggest M). + membrane to presence FIG. the a nuclear major intensity (G0) values S + C2 + cedures (staining mine whether the susceptible Fluorescence activity fluorescence Logarithmically growing Friend virusinduced murine erythroleukemia cells, cultured in the absence and presence of colcemide, were 0 0) C (G1 cells transcriptional method (RNA) tissue Figure 3 shows and green (DNA) culture cell lines and a BM sample (Figure 3D with ALL treated with a deterin 0.1 N HC1 prior to staining II). (Fig. 1B). The distribution of cells within the cell cycle (Fig. 1A and B, solid lines) are reasonably similar for the two methods, though fewer cells (13.9 versus 16.4%) thetic (S), stages of premitotic the cell treated with the hypotonic-citrate ure shows 1C appear cycle the same detergent in 0.1 N with P1 as in Figure The distribution the cell Figure cycle 1A, cycle status, and, the therefore, ity of a cell. distribution followed (staining within information AO staining red which stages 17.0% 0) C F530 (A U) (A U) 0 ‘C U 0) (-) of observed in of the aoo M compart- + regarding procedure fluorescence, relates to RNA to the 0 with various G2 5) by staining method V). to that in the S + syn- pretreated approximately to the second parameter, line, Figure 1A) (7) HCI lB DNA mitotic (M) preparation solution. Fig- cells of cells with the and in the is comparable cells distributed ments. In addition in (G2) transcriptional The major peak to the (Figure 1A) probably cell offers (dotted staining activ- left of the represents a FIG. 2. DNA distribution of FL cells before and after a 6 hr colcemid block. A, green fluorescence (DNA) histogram of logarithmically growing (--) and colcemid blocked (----) cells stained with AO by the one-step procedure at pH 4.0 (method III). B, red fluorescence (DNA) histograms of cells (as in A) stained by the hypotonic-PI staining procedure (13) (staining method IV). Note the apparent lack of increase of cells in the C2 + M phase when treated with the hypotonic citrate solution. STAINING IN CELLS USING ACRIDINE TABLE Morphologic and Staining I Characteristics Hypotonic of Unfixed or Detergent Cells Microscopic Interphase evaluation by Permeable Detergent l’reatment’ - - citrate) Lyse; cells Made Treatment Hypotonic Treatment o.U; 49 ORANGE pH Lyse; nuclei separate in pH 7.() nuclei 4.0 pH Unbroken Unbroken Intact; break under MS.#{176} Stable 3.() pH I.)) Jnbroken agglutinate suspension Metaphases Stainability with AO Broken; chromosomes free or in clumps Broken; chromosomes free or in clumps Stable pH3.0c pH6.0c PHl.0j6.0( Nucleoplasm green green green yellow green yellow green Nucleoli poorly delineated red, when present Metaphase cells present in some, absent red red red red red red remnants, red Metaphase absent red red red red red Cytoplasm Cytofluorometry (AO or P1) Metaphase cells present cells Metaphase present cells Metaphase cells present; 2.4 x higher F,30 in others Cells were made Materials and o Sensitivity sec) or after Cells sity, permeable to Methods. to mechanical application treated the stress stain by (MS.) was detergent evaluated with (Fig. 3A) logarithmically cells while S phase line derived (12) appears levels; the on normal (not shown), G2 from + a to and the line growing proportion M (Fig. patient contain 3C). The blood upper of tetraploid cells distribution of the two have a and “tetraploid” from a patient uniform the be at twice a tively few red (RNA) the G1 cycling cells fluorescence population fluorescence minor population scriptional activity is a of’ cells. A bone marwith ALL appears to DNA distribution (Fig. 3D). histograms only) However, (in this is skewed intensitities of cells (dotted with with line, mitogenic (DNA) pH 3.0 in studantigenic peripheral lymphocytes 3 days in culture is still (RNA) fluorescence and (G0), (DNA) but green C2 + M) in mitosis (Fig. 4C). was ob- served cultures in which this in parallel indicating staining described olds 19 hr prior it percentage dead cells that procedure (9), by setting is a simple (G1 an in S and of cells added fluorescence increased of moder- intensity green in a locus fluorescence (cells An accumulation 4D), tran- at used and there low red (RNA) both (10 human After unchanged red with vortexing 4A) occupy a locus of moderate green (DNA) PHA, with in cells), and red the case a of with increased and cells and intensity. ate green (Fig described indicated. Unstimulated cultures (RNA) presence as normal lymphocytes. cells vigorous (PHA) of cells was toward after as with detergent to staining was response noncycling rela- suggesting increased Fig. 3D). the fluorescence to of fluorescence which overlapping distributions of in control low red lymphocytes appears samples X-100) under the microscope. at pH 1.0, 3.0 or 6.0 (MLC) blood with lymtwo ploidy Triton Cell treatment immediately prior ies of superimposable peripheral population “diploid” row sample higher cell green fluorescence intensity (Fig. 3B). The fluorescence histogram in Figure 3B shows a broad result for EL4 a greater is reasonably human cycling the red the and CEM phoma lower by observing to a drop of cell suspension at pH 3.0 or 1.0 and then stained detergent contains in (0.5-0.1% of a coverslip With FL cultures grown to a high cell denthe majority of cells are in the G, phase of growth treatment to analysis mitoses are (Table the matter vincristine at 3 days (Fig. preserved in I). As previously appropriate to of cells in G0, C,, 5, from such a distribution thresh- quantitate G2 + (Fig. the M and 4E). #{149}0 0 j05 _ FIG. 3. sample with Computer from AO, at a final two-dimensional origin represents cells A, drawn a patient fluorescence text). ALL. concentration distributions the relative as measured Friend scatterplots with leukemia (DNA) C, logarithmically and All cells of 1.1 grown distribution growing to Each a high that appear EL4 cells. cell density as in A. D, a bone marrow (DNA) levels but the red fluorescence suggesting a small population and of dots whose horizontal red (RNA) and green by flow cytofluorometry. cells DNA of C, cell with scatterplot with higher of three cell lines solution The scatter in 0.1 plots is an accumulation B, to represent Compare histograms a detergent method II). and a bone N HCI prior are computer (abscissa) and vertical (ordinate) displacement (DNA) fluorescence intensity, respectively, density. sample from distribution cells RNA were treated 10’ M(staining CEM cells with the cell cycle cells. Note of approximately the two sharp diploid and tetraploid distribution with that peaks amounts of cells marrow to staining generated from the of individual 3 x 10 cells. in the green of DNA grown a patient with ALL. Few cells are seen at elevated of the C, cells appears skewed to increased RNA RNA 50 content. (see to a high green levels STAINING Mixed using for lymphocyte the same were procedure 4B). The fluorescence to that may be (Figure USING analysed after obtained is equivalent PHA cultures and 6 days bution with cultures staining IN CELLS intensity: conditions increases the distriobserved similarly cations. Thus, ence of 5 mM for DNA the green binding dyes by blood ALL are (staining in 0.1 method II). (10). intensity lymphocytes from in There at pH 4.0 (staining at low Specificity of AO: Acridine intercalating staining propidium tion with iodide nucleic staining and teins, several the dye possible per site sites AO per specificity staining dye of precludes is higher than for former are the AO pref- concentrations the must (1, amount be staining. the DNA quan- their F, Corski Rigler (20), to increase Acetylation proteins A, Darzynkiewicz In preparation. of prior interaction several rehydration) MR: of precisely to with steps and is time to stacking dye cells RNA (7) all double involved RNA all DNA in heating have also at agents effect of destroyed cations, ribosomes AO staining that either EDTA (or the presence AL) in as (24). of filled may cell treatment to stainwhile the strength is lower in to change be in achieved temperatures in by (7). with room We chelat- temperature it of is Thus, DNA when in the situ a result just selectively significant at of all RNA in situ, while it does (7). Apparently, the secondary rRNA as 50% stranded it is possible DNA, This EDTA) DNA of pairing moderate almost structure Since in base meta- least to helical. without (i.e., denatures due red double necessary than RNA found at the at 530 nm) stranded RNA prior is single stranded, conformation. cell Since in RNA bonds denature manifested is single helical remains of (488 interactions it that case helical acid-dye dye-dye is ing DNA double on the RNA. of nucleic stacks (1). double of the to use the situ RNA: prop- versus AO nm) in any such versus unique an DNA type are denature tor when in green fluorescence excitation with blue and conformation Z, Sharpless be digestions with the specificity into this (640 cellular the con- be- should in order stain contrast, acids cell (dehydra- It has intercalates upon in ing greatly interaction of DNA be employed and nucleic the be detectable critical AO RNA: results (16) not involves tion, acetylation, suming. 3Traganos of sample. of procedure T, Melamed affinity approach, proposed by of the acetylation reaction amino-residues This are the sites, phosphate the in each A second makes use There this. this approach is hard to impleto maintain a constant molar ratio for titated pro- Since AO at low DNA available the for stained 16). However, ment, since doing (26). binding erentially be precluded. of may chromasia mostly of control to assure differentially AO light; electrostatic to the acids. staining interaction with spe- result is especially of acid nm involves the use of AO at low that is at a low molar ratio of intercalating of must binding electrostatic the to Namely, in its interacto its positive polyanions, ways One approach concentrations, AO acids, between that dye nucleic interacts electrostatically (1). Therefore, to obtain of nucleic interactions erty with macromolecules all nucleic Differential than other bromide, acids specific ethidium or mithramycin acids. Due AO also polyanions cific of nucleic orange is less dyes, such as the however, that are required Denaturation DISCUSSION the appears virtually and This (Nat or Mg2) AO (10’ M) for to contain large quantities of polyanions glycosaminoglycans) are stained. (i.e., pH 5). charge, other is AO of acids and fluorescence known in green treatment (14) of staining. deter- cations with staining nucleic stressed, nucleases between AO and if cells are stained in the presence of cells may be stained in the presMgC12 or 0.1-0.15 N NaCl (6, 9). sites than tween a patient increase after to when with anionic impeded due DNA one-step is a 2.4-fold intensity 5 shows (F,30) in 51 Under conditions where are in excess, competing other sites Figure III) or first treated HC1 prior to staining N fluorescence binding intercalation stained method (Fig. available fluorescence peripheral gent of intercalating AO with Treatment of cells at low known to extract histones, number ORANGE A highly specific interaction nucleic acids also is obtained in equilibrium with the dye culture analysed3. Staining pH under ACRIDINE in the divalent of isolated for RNA cells cell of case conditions versus intact chelation in are is differential cells are pretreated fulwith citrate), or when they are stained of these chelating agents. concentration: The differential staining other significant of double in fac- versus MLC-6 Days ,-‘ r - i. - r:- ... 1 - I : .4 #{149}1 / 1TT --- - - I 0 100 50 PHA - 50 3 Days --, ;fr. .-, .:* t’ ..-.. I I ‘: . ‘ , - - - - - - - SI tUl 00 FIG. 4. DNA and RNA distribution in control detergent at pH 3.0 and then stained with AO and red (RNA,----) fluorescence histograms and stimulated (staining Method of human peripheral 52 human leukocytes. The I).A, scatterplot (as in blood leukocytes cells Fig. cultured were 3) green for treated with (DNA,--) 48 hours in the STAINING IN CELLS USING 5) 0 x (32 0)1 C I) (77 0 ‘C Fluorescence (A U) Difference in green (DNA) fluorescence of cells treated at pH 4.0 or in 0.1 N HC1 and with AO. Peripheral blood leukocytes from a with ALL were stained by the one-step (III) intensity stained patient AO method 0.1 N or first (-) HC1 (II) followed treated by with staining detergent with optimal --). all nucleic As we the same. acids have is the shown concentration sites dye Ref. that are F,30 approaches staining is done in at a the at presence of the N NaCl cations. With the optimal between 5 x 10’ varies Considering the concentration binding which of absence of nucleic concentration DNA phosphate Cell makes per sample an cation, of low molar the single as and excess the of fixed cluding centrifugation, cells involve some cell cells on the other rameter cells to the can (see for help 18) of nucleases Hypotonic introduced treatment: a technique propidium to break nuclei. according not have integrate dye fluorescence analysing molar or treated ment. vides AO/ DNA (13) tion on the since some or all Loss of cytoplasmic and cell types Krishan (13) of nonfixed a hypotonic thus makmethod staining microscopy, Table colcemid or used to obtain in G2 escape ymca cells treat- method proof cellular of cells may I). cultures” of metaphase the hypotonic M cells disor within the remobserved under “synchronized be is of such treatthese cells do while the hypotonic and uniform staining proportion the they totally chromosomes (as with it cannot assess the hypotonic treatauthor, “results in a subpopulation detectable using AO stain- some dye. The uniform clumped membrane when Thus, rapid dead iodide using membranes, cell or UV double pa- from to a nuclear envelope, leaving either isolated cultures This living (13). Unfortunately, mitotic cells; since chromosomes of the cell informa- uptake. controls However, to the some nants alkaloids, was not of nonfixed provides Recently, for staining phase an Staining classify as of staining. diploid ment, inand of dye transport into the cell in lysosomes (17). It does staining of nucleic acids or specificity cells with treatment steps, consuming hand, or the Unfortunately, differentiate Ref. nude- evaluate several loss. in vivo Thus, for agents optimal use (7). is time living to chelating to requires may These exogenous possible on (7). only to staining AO However, at it staining related not also of AO optimum to be to chelating or of M. 3 x 10-’ M and ratio near 0.4 (26). bases on 10’ versus appears The fixation: 4 x (8, 26). acids as and phosphate permeability Cell of isolated nuclei” ment also lyses concentraand conditions any the Since competing (AO/DNA staining stranded but established acetone ing cells permeable to the rapid and gives an excellent, 5 mM MgCl2 concentration is always those differential AO: number site under the cell there (6). plateau. cations (Mg2 or Nat), this optimal tion of AO depends on the concentration the affinity 0.10-0.15 which saturated range in the by a plot of F,30 versus 26), and is indicated concentration change concentra- previously, is intercalation This may be determined AO concentration (see per which tion were permeable, the the DNA alcohol: AO, approximately dye nearly ratio) fully and fluorescence intensity for the C0/C, by 2.4 times (32.1 to 77.8) after at low pH, the coefficient of variation (CV) of’ AO. in (-- vs. in based on the rate and its accumulation not permit selective stranded tion AO in AO the mean increased treatment remained by intensity 5. single are RNA specificity ci) C.-) While cells prefixed ases, (I) FIG. of cells cells 53 ORANGE ability agents cv491\ 0V48 C 8) ACRIDINE informaM phase, + detection. components, including absence of stimulant. Note moderate green (DNA) and low red (RNA) fluorescence. B, mixed lymphocyte culture stained as in A. Cells with increased red (RNA) fluorescence (C,) or increased green (DNA) and red (RNA) fluorescence (S + C2 + M) can be observed in addition to C0 cells. Cells with decreased green fluorescence represent dead or dying cells. An additional population to the left of the C0 cells has been thresholded out for clarity and will be discussed in a future publication3. C, human peripheral blood leukocytes cultured for 3 days in the presence (B). D, cells accumulation cence histogram. fluorescence chronous of PHA (9). The distribution of cells is very similar to that observed in mixed lymphocyte cultures as in C in which 0.2 og/ml vincristine was added 19 hr prior to analysis at 3 days. Note the in the C2 + M phase of the cell cycle in both the scattergram and the green (DNA) fluoresD, lymphocytes cultured with PHA for 3 days. The thresholds for red (RNA) and green (DNA) cultured of cells are cultures masked were to discriminate used to identify cell the subpopulations. positions Control experiments of the subpopulations and with thresholds, blocking agents as presented or syn- (9). 54 TRAGANOS RNA, ment ET the also precludes using the hypotonic treatfor RNA staining of interphase cells. Treatment sure with of cells known brane to detergent at detergents at to induce disintegration and subsequent methods of isolation phenomenon. lyse when also noted are with detergent the nucleoli clei from lysed cells stain I), suggesting that RNA red does levels of F>,00 differ from membranes, which stains red, but are made the cytoplasm while (Table There the permeable procedure, by the with have do not ways detergent. dye (AO P1) denaturing agent (EDTA) are single solution; the pH of that tained at 4.0. This since we promise, at the pH below interaction acids at pH the above a comwith AO the specificity of dye and nucleic cells lyse. Unfortunately, although cells do not lyse 4.0, they do appear to be fragile and break 4.0, at pH down after additional mechanical pipetting or mixing. Thus, the one-step seems to coefficient the for possible ing during the flow cell mechanical is like the the simplest resolution (the it requires lysis under vigorous fact that and lowest careful forces con- of shear- raised. retains stress, procedure, at pH are and at higher pH. During the concentration of detergent dye solution is added, and Under such -treatment its integrity and resistence yet it becomes nucleic permeable It is the to to cells from absence (i.e., in saline), of for a several Triton acids is carried therefore which as a result (e.g., X (0.02- out at pH unlikely may have of RNA that been 3.0 any solubilized denaturation) are lost due to their insolubility under acid conditions. When detergent treatment is carried out 0-4#{176}Cat pH nous 3.0 or below, nucleases, appear which to be any may effect have precluded. We at of endoge- been released, have observed that in experiments in which the time of detergent treatment was varied from 1-5 mm no alteration in the mean fluorescence intensities could shown) be observed suggesting cationic Most for the cell that nucleic and/or DNA is acid-soluble ble, stainable types stage types studied (not acids are not lost or nucleolytic versus total intercalating unavailable, proteins. DNA dyes being The portion and in some cell of genome activity above, stances extent varies types (26). it would appear to be advantageous DNA rather than in attack. DNA: isolated with (10, 20, masked the 26). by of the availabetween cell is related Considering to the the under some circumto stain all nuclear only the unmasked portion, thereby providing a more constant (although still relative) measure of DNA per cell. By this approach, the differences in chromatin composition or conformation may be eliminated, which may ties the cells 3.0 or lower in their treatment below. be DNA in a cytofluorometer. two-step with detergent then are stained staining step the drops as the buffered the pH membrane the stress despite is best of variation) trol In treated procedure offer or all directly serum. Serum promembranes against ofcells concentration for X-100, make taken Only a portion of DNA in situ or chromatin is available for interaction RNA into a is main- staining to are either through “leakage” Staining of unmasked cells a one-step and represents that 3.5 decreases between the while green combined solution procedure found to AO deeply disintegrate. to stain In or in detergent ruptured fluoresces sufficient they chosen (Triton is adequate. also permeable and nucleoli nucleoplasm I). Cells in mitosis are two possible made seen lower Detergent nu(Table out of those nucleases. detergent minimal Therefore, 0.04%) them. In these preparations of broken cells (plus isolated nuclei) mitotic cells are lost, and the intensity of F>,00 and proportions of cells at various yet when suspensions fold above AO leak was v/v), staining to be lysed. with not parallel, unbroken cell preparations. We observed that cells treated at pH 4.0 and below do not 0.07-0.1% detergents. of isolated of the cultures containing 10-15% teins markedly stabilize cell on this at pH as to exogenous experiments permeable by phasethat cells all cells appeared that is memVarious experiments, and I), as well concentration these pH cell based controlS fluorescence (Table treated 4.0; at neutral pH We cell in UV neutral dye The Expo- pH: of the lysis. of nuclei Indeed, we observed by interference-microscopy low AL. As cells gent, particularly content helpful of cells or of different described with 0.1 removes proteins and intercalating types above, N HC1, histones in the and increases the sites reacting fold. Despite this rather remain in relatively and do not assaying genome the activi- in the same sample. treatment of nonfixed cells condition when of different presence of deterother acid soluble maximum with harsh good aggregate. dyes number by treatment of 2.4the morphologic We want to STAINING stress, the that however, DNA the acid to prevent treatment should cold (0-4#{176}C) (25). Treatment of cells significantly with change AO. Therefore, regarding sired to (i.e., tion, with genome detergent step procedure of the correlate at pH staining cells with of analysing on a normally required is avoided, in ploited tion this on the (21, for cell hand With The AO staining plied to cell cycle perturbed mia, of and Since fixation produced and on mea- additional parameters several apcell cycle with leuke- blood leuko- cell-cell interaction be pointed procedures have lymphoid cells. those containing of fibroblastic out, been however, tested Other Exposure dure of cells for making digestion to detergent is a more cells treatment. method be Kurland J, Traganos MAS: In preparation. at low permeable or care- pH, as procethan is the Therefore, we used in conjunction suggest with F, Darzynkiewicz the CITED MK: JD, Aggregation Proc Natl Melamed F, Sharpless MR, Acad of dyes Sci USA Darzynkiewicz T, Good lymphocytes and staining Z, RA: Quantitation by flow phytohaemagglutinin methods acid and protein chem Cytochem Crissman minutes. cytofluorime- by flow cyto- for analysis of deoxyribonucleic in mammalian 24:64, 1976 HA, Tobey RA: Science 184:1297, cells. Cell cycle 1974 J Histo- analysis in 20 6. Darzynkiewicz Z. Traganos F, Sharpless T, Melamed MR: Thermal denaturation of DNA in situ as studied by acridine orange staining and automated cytofluorometry. Exp Cell Res 90:411, 1975 7. Darzynkiewicz Z, Traganos F, Sharpless T, Melamed MR: Conformation of RNA in situ as studied by acridine orange staining and automated cytofluorometry. Exp Cell Res 95:143, 1975 8. Darzynkiewicz Z, Traganos F, Sharpless T, Melamed MR: DNA denaturation in situ. Effect of divalent cations and alcohols. J Cell Biol 68:1, multiparameter 73:2881, to deter- advantageous Nager of fluorometry. II. Comparison with “C-thymidine incorporation. Clin Immunol Immunopathol, 5: 326, 1976 4. Crissman HA, Oka MS. Steinkamp JA: Rapid on especially large amounts of polyanions or epithelial origin, require above, hypotonic that this types, Robin preparation 1976 these predominantly cell ful controls utilizing nuclease mine staining specificity. discussed that Miss the Wolf 9. Darzynkiewicz Melamed MR: systems. It should or hypo- try. Clin Immunol Immunopathol 4:209, 1975 3. Braunstein JD, Melamed MR, Sharpless TK, Hansen JA, Dupont B, Good RA: Quantitation of lymphocyte proliferative response to allogeneic 5. analysis. patients to thank in to polyanions. 1959 Braunstein cells informadistribu- or antigenic stimulation. Infrom RNA staining is curto assess the cytostatic efin 2. DR. of transformed metais ex- cell Bradley bound 45:944, counterstaining agents, wish assistance Traganos (often The which authors her LITERATURE 1. permeability of peripheral cytes to mitogenic formation obtained rently being used fects used. and DNA in simple, fast cell systems channels, be obtained from response fixation manuscript. staining or a single fluorescence) various specimens the differentia- a two-step pH 3.0, The for procedures have been analyses of tissue culture by P1, mithramycin, to cell ACKNOWLEDGMENT is de- fluorescence ratios may and bromide, alternative not DNA independent and cell cycle offering (ethidium an information cell (green dyes reacting activity (red fluorescence) the addition of pulse width thereby analysis does of DNA makes identification lines treatment. are required. with AO circuits on both to cytoplasmic 23) tonic or centrifugations provides levels one as membrane system and ploidy transcriptional the other. suring nuclear for cell loss) associated in unnecessary tion as to etc.) scale. no washings resulting chromasia other 3.0-4.0 55 ORANGE of 4.0 should be of RNA AO offers a a variety of microculture ACRIDINE in stainable at USING denaturation when etc.) CELLS be performed it with activity, treatment Simultaneous even pH extent in cases quantity unfixed means at the IN Z, Moore Z, ‘I’raganos Lymphocyte analysis. Proc F, Sharpless T, stimulation. A rapid Natl Acad Sci US, 1976 10. Darzynkiewicz Z, Traganos F, Sharpless T, Friend C, Melamed MR: Nuclear chromatin changes during erythroid differentiation of Friend virus-induced leukemic cells. Exp Cell Res, 99: 301, 1976 11. Dukes CD, Parsons JL, Stephen CAL: Use of acridine orange in lymphocyte transformation test. Proc Soc Exp Biol Med 131:1168, 1969 12. Kapaln J, Shope TC, Peterson WA Jr: EpsteinBarr-virus-negative human malignant T cell Lines. J Exp Med 139:1070, 1974 13. Krishan A: of mammalian staining. Rapid J Cell cell flow cytofluorometric cycle by propidium Biol 66:188, 1975 analysis iodide 56 TRAGANOS 14. LePecq JB: Use of ethidium bromide for separation and determination of nucleic acids of various conformational forms and measurement of their associated enzymes. Methods Biochem Anal 20:41, 1971 15. LePecq JB, Paoletti C: A fluorescent complex between ethidium bromide and nucleic acids. J Mol Biol 27:87, 1967 16. Lerman LS: The structure of the DNA acridine complex. Proc Natl Acad Sci US 49:94, 1963 17. Melamed MR, Adams LR, Traganos F, Kamentsky LA: Blood Granulocyte staining with acridine orange. Changes with infection. J Histochem Cytochem 22:526, 1974 18. Melamed MR, Kamentsky LA: Automated cytology. Internatl Rev Pathol 14:205, 1975 19. Ralph P: Retention of lymphoid characteristics by myeloma and theta lymphomas. J Immun 110:1470, 1973 20. Rigler R Jr: Microfluorometric characterization of intracellular acridine 1,1966 nucleic orange. Acta acids Physiol and nucleoproteins Scand 67:suppl by 267, ET 21. 22. 23. AL. Sharpless TK, Melamed MR: Estimation of cell size from pulse shape in flow cytofluorometry. J Histochem Cytochem 24:257, 1976 Steinkamp JA, Crissman HA: Automated analysis of deoxyribonucleic acid, protein and nuclear to cytoplasmic relationships in tumor cells and gynecologic specimens. J Histochem Cytochem 22:616, 1974 Steinkamp JA, Hansen KM, Crissman HA: Flow microfluorometric and lightscatter measurement of nuclear and cytoplasmic size in mammalian cells.J Histochem Cytochem 24:292, 1976 24. Tal M: Metal ions and ribosomal conformation. Biochem Biophys Acta 95:76, 1969 25. Traganos F, Darzynkiewicz Z. Sharpless T, Melamed MR: Denaturation of deoxyribonucleic acid in situ. Effect of formaldehyde. J Histochem Cytochem 23:431, 1975 26. Traganos F, Darzynkiewicz Z, Sharpless T, Melamed MR: Cytofluorometric studies on conformation of nucleic acids in situ. I. Restriction of acridine orange binding by chromatin proteins. J Histochem Cytochem 24:40, 1976