Fixation-associated quantitative variations of DNA fluorescence observed in flow cytometric analysis of hemopoietic cells from adult diploid frogs

0 1990 Wiley-Liss, Inc. Cytometry 11:676-685 (1990)

Fixation-Associated Quantitative Variations of DNA Fluorescence Observed in Flow Cytometric Analysis of

Hemopoietic Cells From Adult Diploid Frogs Hiroko B. Holtfreter' and Nicholas Cohen

Department of Microbiology and Immunology, The University of Rochester School of Medicine and Dentistry, Rochester, New York 14642

Received for publication November 22,1989; accepted April 7, 1990

We have examined, by flow cytometry, the apparent DNA content of frog blood cells that had been fixed with either 50% ethanol, 70% ethanol, or 66% methanol, before being stained with either mithramycin, propidium iodide, or Hoechst 33258. After 50% ethanol fixation, regardless of the dye used, the DNA content of the hemopoietic cells appeared unimodal, but after either 70% ethanol or 66% methanol fixation, it appeared bimodal. Cell sorting revealed that the lower and upper modes are represented by erythrocytes (RBCs) and leukocytes (WBCs), respectively. In amphibians, the chromatin of metabolically inactive RBCs is highly condensed relative to the chromatin of

metabolically active WBCs. The bimodal distribution of DNA contents seen with 66% methanol and 70% ethanol, but not 50% ethanol, seems to reflect this disparity in the degree of chromatin condensation existing between the RBCs and WBCs. This, in turn, implies that the accessibility of fluorescent DNA dyes to the chromatin of fixed frog hemopoietic cells, especially of RBCs, can be affected by the concentration of alcohol used for their fixation.

Key terms: DNA fluorescent dye accessibility, fixation artifacts, frog hemopoietic cells, erythrocyte chromatin compaction, ploidy

We and others have been using diploidhiploid chimeras to investigate the developmental origin of hemopoietic cell in the frogs, Rana pipiens and Xenopus laeuis. Chimerism was established prior to larval life by fusing defined regions of diploid (2N) and triploid (3N) tailbud stage embryos (3,11,22). In principle, the ploidy of blood cells in chimeric tadpoles or adults should indicate the embryonic site(s) of that cell type. During the course of some control studies that involved different fixation protocols, we noted that the amount of DNA in RBCs and WBCs of normal (i.e., non-chimeric) 2N or 3N perimetamorphic tadpoles, froglets, and mature frogs appeared to differ considerably as if the RBCs and WBCs from a single animal were of a different ploidy. These observations compelled us to investigate the possibility of fixation artifacts, and to es- tablish a reproducible fixation protocol for flow cytometry that allows fluorescent DNA dyes to bind stoichiometrically to the chromatin of all hemopoietic cells (i.e., independent of their developmental stage or degree of chromatin compaction) such that the dye bound to the chromatin accurately reflects the cells' ploidy.

MATERIALS AND METHODS Materials

Non-chimeric diploid adult frogs, Rana pipiens (J.M. Hazen and Co., Alburg, VT) and Xenopus laeuis (South African Snake Farm, Capetown, South Africa), were used. In addition, a few non-chimeric 3N adult Xenopus laeuis (9,24) were also used.

Methods Cell suspension medium (CSM). Hemopoietic

cells were suspended in Leibovitz-15 medium (L-15) (Gibco, Grand Island, NY) that was diluted to the os- molarity of adult frog serum (220 mOsm) and supple- mented with 1.25 x lop5 M HEPES buffer, 100 U/ml penicillin, 100 kg/ml streptomycin, and 2% fetal bovine

'Address reprint requests to Dr. Hiroko B. Holtfreter, Box 672, Dept. of Microbiology and Immunology, University of Rochester Med- ical Center, Rochester, NY 14642.

DNA DYE BINDING TO FIXED FROG BLOOD CELLS 677

serum (FBS). Peripheral blood was collected in CSM that contained 2 U/ml heparin.

Preparation of cell suspensions. Hemopoietic cells examined were from peripheral blood (Pbl; collected from heart), and from the spleen (Spl), kidney (Kid), and liver (Liv). Thymocytes were not examined owing to the virtually complete involution of the adult frog thymus. Only a portion of the liver was excised. Each organ excised was rinsed thoroughly in CSM and then teased apart with watchmaker’s forceps in a small plastic dish containing 1-2 ml of cold CSM. Free cells suspended in the medium were collected in a 15ml polystyrene centrifuge tube. The remaining tissue fragments were again teased after they were flushed with a small amount of fresh CSM. Free cells resulting from this rinse were added to the tube. Cell suspensions from kidney and liver were washed once or twice with cold CSM. The final volume of cell suspension per sample was adjusted to 3-6 ml and aliquoted into two or three tubes to examine effects of different fixation procedures on cells from the same tissue sample. The number of cells per tube was adjusted to be not more t h a n 3 x 106/ml CSM.

Fixation. The various fixation procedures employed were designated as follows: 66% methanol (MeOH) fixation (two parts of ice-cold MeOH added to one part of a cell suspension); 70% ethanol (EtOH) fixation (2.5 parts of ice-cold absolute EtOH added to one part of a cell suspension); 50% EtOH fixation (ice-cold absolute, or 95% EtOH, added to a cell suspension in equal amounts). Fixatives were gently poured into cell suspensions which were immediately vortexed for several seconds to achieve uniform fixation of the cells.

Solutions for stainings. The fluorescent dyes used for DNA stainings were mithramycin (MI; Miles Phar- maceuticals, West Haven, CT), propidium iodide (PI; Sigma), and Hoechst 33258 (HO 258; Calbiochem, Beh- ring Diagnostics, La Jolla, CA). These dyes were se- lected because of their different binding characteristics to nucleic acids. PI intercalates between the base pairs of both DNA and RNA (6,27), whereas MI interacts with only DNA and i t does not intercalate. MI preferentially binds to G-C regions (6,271, whereas HO 258 preferentially binds to A-T regions of DNA a t neutral pH (16,23); HO 258 also binds to RNA at pH 3 (16). Solutions used for the stainings were prepared as follows: MI solution: 2.5 mg of MI was dissolved in 6.25 ml of 50 mM MgC1, and 18.75 ml of APBS. PI solution: 5 mg of PI was dissolved in 100ml of APBS. Ribonu- clease (RNase) solution: 10 mg of RNase (Calbiochem) was dissolved in 10 ml double-distilled water by heat- ing it to a boil and then adjusting its pH to 7.0-7.4 with 0.05 M NaOH. This solution was aliquoted in plastic vials and kept at -20°C until it was used. VindelGv’s PI solution (26) was also used with the following modifi- cations: 1) with the non-ionic detergent, Nonidet P-40 (NP40) (PI solution 200 pl, RNase solution 60 pl, 0.1% NP40 in APBS (v/v) 60 pl and APBS 280 p1) or 2) without NP40 (PI solution 200 p,l, RNase solution 60

pI, and APBS 340 11.1). HO 258 solution: 100 pM solution in APBS was used as a stock solution and stored at -20°C. Immediately before its use, it was diluted to 3 pM with APBS, pH 7.03.

Staining procedures. MI staining (4,5): Fixed cells were rinsed once or twice in APBS (2 ml per sample tube) and resuspended in the MI solution. The cells in the dye solution were either analysed by flow cytometry a Sew hours later, or kept in the dark at 4°C overnight (occasionally for 2-3 days) before analysis. PI staining (5,18,26): Fixed cells were rinsed once in APBS and resuspended in 2 ml of APBS. Digestion of cellular RNA was carried out a t 37°C for 30 min by the addition of 200 pl of RNase (100 pl RNase solution per 1 m of APBS). The cells were then washed with APBS and resuspended in cold APBS, to which the PI solution (half the amount of the APBS) was subsequently added. They were stained at 4°C for several hours, or more frequently, for overnight to a few days. Modified Vindelflv’s PI staining was also employed for a few sets of specimens: Fixed cells washed once in APBS were stained at 4°C in the PI solution containing either RNase or RNase plus NP40. The flow cytometric analysis of these modified Vindelov’s PI-stained specimens was carried out not later than 1-3 h after the staining (in the dye solution). HO 258 staining (1,16): The cells, fixed in 70% EtOH or 50% EtOH, were rinsed once in APBS, resuspended in 3 pM HO 258 solution, and refrigerated overnight prior to cytometric analysis.

Cells were pelleted between each step of the foregoing protocols in a refrigerated centrifuge for 5 min at 300g. Cells pelleted a t the last step of every staining procedure were resuspended in an amount of the solution (either a staining solution or distilled water) to a final concentration of approximately 1 x 106/ml (for analysis by flow cytometry).

Flow cytometry. Immediately before the flow cytometric analysis, the stained cell suspensions were vortexed, drawn three times through a 22-gauge needle attached to 3 ml stylex syringe, and filtered through 45 pm nylon mesh to remove cell clumps. The nylon mesh was wetted with APBS before its use to prevent significant cell loss. DNA analysis and cell sorting were carried out on an EPICS V multiparameter flow cytome- ter/cell sorter (Coulter Electronics, Hialeah, FL) with a 5 W UV-enhanced argon-ion laser (Spectra Physics, Mountainview, CA, Model 2025) tuned to an excitation wavelength either of 457 nm at a power of 200 mW (for the analysis of MI-stained cells) or 488 nm a t a power of 500 mW (for the analysis of PI-stained cells). Green (515 nm-560 nm) or red (> 610 nm) fluorescence emission was collected depending on the dyes used. For the analysis of HO 258-stained cells, the laser was tuned to the triplet of near-UV lines from 351.1 to 363.8 nm at a power of 100 mW, and blue fluorescence emission was collected above 418 nm. Cell size was estimated by low-angle light scatter pulse-width time-of-flight (LSPW) with an outboard circuit module (20) recently modified to allow for real-time subtraction of the laser

678 HOLTFRETER AND COHEN

beam width by using a high-precision biased amplifier. The LSPW scale was calibrated with a mixture of polystyrene microspheres with the diameters of 5.08 pm and 9.6 pm (Duke Scientific Corporation, Palo Alto, CA). A mixture of 5.08, 9.6, and 20 pm-diameter microspheres was also used occasionally for the samples of Rana cells. These calibrations remained stable throughout the duration of each experiment (3-4 h). Data were acquired on an 8086-based microcomputer (MDADS, Coulter Electronics). Cells were sorted according to user-specified windows on either one or two parameters.

Cytospin preparation. RBC and WBC types were identified morphologically. Cell (living or fixed) suspensions at a concentration of 1-5 x lo5 cellsiml were cytocentrifuged (Shandon-Southern) on microscope glass slides: (200 pl per sample-chamber for 5 min a t 600 RPM). Slides with spun cells were air-dried (living, spun cells were then fixed in absolute MeOH and air- dried again), stained by the double staining of Delafield haematoxylin and eosin Y, dehydrated through a series of EtOH and xylene, and then mounted by caedax (Merck) under a cover glass for microscopic examination. WBC differentials were not performed. Sorted cells were collected in 35 x 10 mm Falcon dishes (#1008) which contained 200 pl of 10% FBS in double-distilled water. They were either pipet- ted from the dish directly into the specimen chamber or pelleted once in a microfuge tube (1.5 ml) prior to cy- tocentrifugation and then resuspended in 10% FBS. The slides with spun cells were then processed according to the preceding protocol.

RESULTS 66% MeOH or 70% EtOH Fixation Combined

With MI, PI, or HO 258 Staining MI staining. Flow cytometry of the diploid speci-

mens that were stained with MI solution after fixation in 66% MeOH (MM) or 70% EtOH (7EM) revealed two populations of cells that seemed to differ in their DNA contents. That is, the single-parameter histograms of integrated green fluorescence (IGFL) displayed clear bimodal distributions (Fig. 1, first and third columns). The “secondary” peaks occurred, in most cases, at less than twice the values of the “first” peaks, indicating that the different peaks did not reflect GI and G, + M peaks of cycling cells (see Table 1). Characteristics of this bimodal distribution are better illustrated in two- parameter histograms (two-dimensional isometric con- tours and three-dimensional isometric displays of IGFL vs. LSPW; IGFL and LSPW reflect relative DNA contents and sizes of the cells measured, respectively). The cells with the higher values of IGFL occurred almost exclusively at lower ranges of LSPW, whereas the cells with lower values of IGFL occurred a t very broad ranges, reaching quite high scales of LSPW (Fig. 1, second and fourth columns). In other words, those histograms demonstrated that the cells with the higher values of IGFL consisted mainly of small cells, whereas

those with the lower IGFL values were the cells of di- verse sizes (from extremely large to small ones that were comparable to those seen at the higher IGFL).

Sortings these two populations of cells decisively demonstrated that the cells with the lower values of IGFL were RBCs, whereas those with the higher IGFL were nearly all WBCs (Fig. 2A-C). Inconspicuous presence of the second peaks (with higher IGFL values) in the histograms of Pbl mirrored the fact that the majority of the Pbl samples contained WBCs in very minor proportions.

A comparison of cells from the same organs that were fixed in 70% EtOH (7EM) and 66% MeOH (MM) and analysed at the same instrument gain settings revealed that although IGFL peak values of the WBCs in the two samples were nearly the same, peak values for RBCs were not (Table 1, Samples RA2 and XE7). Spe- cifically, the peak values of the RBCs in the specimens of 7EM were lower than those of the RBCs in the specimens of MM (compare columns two and three of MI staining in Table 1). This implies that accessibility of MI to the chromatin of RBCs, but not of WBCs, was influenced by the fixatives. Student t-tests revealed that the W/R ratios (IGFL peak value of WBC vs. that of RBC) differed significantly between the paired sets of MM and 7EM samples in both Rana (0.001 < P < 0.01) and Xenopus (0.001 > P < 0.01). That is, the binding of MI to 70% EtOH-fixed RBCs was significantly less than its binding to 66% MeOH-fixed RBCs.

A morphological observation that fixation by 70% EtOH caused more shrinkage of RBCs than 66% MeOH is exemplified by the following. According to microscopic measurements of Xenopus RBCs, a mean 20.4 % 0.2 pm of the long axis of living RBCs decreased to 11.7 ? 0.2 pm (43% reduction) and to 15.1 t 0.2 pm (26% reduction) after they were fixed in 70% EtOH and 66% MeOH, respectively. The difference of these two means of differently fixed samples was highly significant (P <0.001). How or to what extent this shrinkage is relevant to the accessibility of MI to the chromatin of RBCs, however, remains speculative. The shrinkage of RBCs was also revealed in the two-parameter histograms in that the 70% EtOH-fixed RBCs were distrib- uted over much lower scales of LSPW, in comparison to the 66% MeOH-fixed RBCs, whereas distribution scales of the 70% EtOH-fixed and 66% MeOH-fixed WBCs were the same (Fig. 1, second and fourth columns).

The differences of means of W/R ratios between non- paired sets of MM samples (n = 28) and 7EM samples (n=23) were highly significant (P <0.001, data not shown). Moreover, the t-test revealed that differences in the means of WIR ratios between two organs such as Pbl and Kid, and Pbl and Liv were significant, although differences of the means between Spl and Kid, Spl and Liv, and Kid and Liv were not. In the case of Pbl and Spl, the difference of their means differed significantly in the 7EM set, but not in the MM set. Whether these differences (or similarities) of W/R ragios signify “ac-


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Relative DNA Content

FIG. 1. “DNA distributions” of adult Xenopus hemopoietic cells: Bimodal distributions obtained from the cells which were stained with MI, after they were fixed in either 66% MeOH (MM set of first and second columns) or 70% EtOH (7EM set of third and fourth columns), and unimodal distributions resulted from the fixation of the cells in 50% EtOH which were then stained in modified Vindelprv’s PI solution without NP40 (5EP set of fifth and sixth columns). A, B, C,

and D: Peripheral blood (BMM, B’IEM, B5EP), spleen (SMM, S7EM, S5EP), kidney (KMM, K7EM, K5EP), and liver (LMM, L7EM, L5EP), respectively. Total number of cells counted in each sample was 50,000 Samples of the spleen, kidney, and liver of the sets MM and 7EM were sorted according to the “windows” specified with bars in the figures AL (BL) and AU (BU) indicate the lower and upper limits of a sorting window A (B), respectively. CV = coeficient of variation

tual conformational differences (or similarities)” of the chromatin of the hemopoietic cells in each organ at the time of fixation is unknown. It may be relevant that occasionally the samples of Kid and Liv, and less frequently the samples of Spl and Pbl, produced three IGFL peaks. The first peak seen at the lowest scales of

IGFL was of RBCs, and the peaks of the second and third were of WBCs. The cells constituting the second and third peaks were similar in size according to their distribution ranges of LSPW histograms, although sometimes the cells of the third peak were smaller than those of the second peak. The occurrence of this extra


Table 1 Ratios of W peaks Vs. R peaks (W/R) of Specimens Stained With Either Mithramycin or Propidium

Zodide After Fixation in 66% MeOH or 70% EtOH“

66% MeOH (MM) 70% EtOH (7EM) 66% MeOH (MP) 70% EtOH (7EP) Samples W peak/R peak W peak/R peak Samples W peak/R peak W peaWR peak RA2 Blood 117/85 = 1.38 117/65 = 1.80 RA2 Blood 105/75 = 1.40 98/70 = 1.40

Spleen 109178 = 1.40 108168 = 1.59 Spleen 104/75 = 1.39 103170 = 1.47 Kidney 117196 = 1.22 118175 = 1.57 Kidney 108181 = 1.33 105169 = 1.52 Liver 106/77 = 1.38 102/60 = 1.70 Liver 92/65 = 1.42 89/57 = 1.56

XE7 Blood 96/57 = 1.68 125150 = 2.50 XE3 Blood 116/74 = 1.57 110170 = 1.57 124/70 = 1.77

Spleen 102164= 1.59 101151 = 1.98 Spleen 111/74= 1.50 126/83 = 1.52 145/83 = 1.75

Kidney 98/57 = 1.72 100149 = 2.04 Kidney 111/79= 1.41 107/75 = 1.43 123175 = 1.64

Liver 102161 = 1.67 102/48 = 2.13 Liver 108/76 = 1.42 11 1/75 = 1.48 121/61= 1.98 120148 = 2.50 145175 = 1.93

“W: White blood cells. R: Red blood cells. MM, 7EM: MI staining in combination with 66% MeOH and 70% EtOH, respectively. MP, 7EP: PI staining in combination with 66% MeOH and 70% EtOH, respectively. RA2, XE7, and XE3 represent experiments performed on three different individuals. Each of the four sets of data represents an experiment carried out under identical instrument gain settings within a few hours. Therefore, peak values within, but not between, data sets can be compared.

peak of WBCs suggests that evidently a certain population of WBCs bound MI more than the remaining populations of WBCs. In a few instances, Pbl contained a population of WBCs whose IGFL peak value was much higher than that of the WBCs in the remaining organs derived from the same animal, but this peak value often matched the third (WBC) peak value (XE7, 7EM; Table 1). The cause of this disparity of the dye binding among the WBCs is unclear. It is interesting, however, to note that the WBC’s extra peak appeared in every organ sample of the 7EP series (e.g., specimen XE3 in Table l), whereas it did not appear in any of the samples in MP series. Whether these extra peaks of WBCs observed here correspond to the peak of “spu- rious aneuploidy” observed by Cunningham et al. (7) with “non-purified” human peripheral blood that was fixed in 70% EtOH and stained with MI is unknown.

For specimens fixed in the same way, profiles of the bimodal distributions of DNA in the hemopoietic cells, including the appearance of an extra WBC peak de- scribed above, were not affected by the duration of either staining (1 h to 4 d) or fixation. When some specimens analysed 2-7 d after fixation were reexamined 2 or 3 months later, virtually identical DNA distribution patterns were seen.

P I or HO 258 staining. Results from the flow cytometry of specimens stained with either PI or HO 258 were very similar to those obtained from aforemen- tioned specimens that were stained with MI. However, accessibility of these two dyes to the chromatin of RBCs appeared to differ from that of MI. Namely, in contrast with the results obtained from the comparable MI staining, the uptake of PI by the chromatin of the 70% EtOH-fixed RBCs (7EP) and 66% MeOH-fixed RBCs (MP) was equal (see fifth and sixth columns in Table 1; 0.05<P<O.1) according to their W/R ratios. The means

of W/R ratios between non-paired MP and 7EP samples were also similar (0.05<P<O.1).

W/R ratios of the specimens that were fixed by 70% EtOH and then stained with HO 258 (7EH) were greater than those of the identical specimens which were fixed by 70% EtOH but stained with either MI (7EM) or PI (7EP). The difference of W/R ratios between the samples of 7EH and 7EM and those of 7EM and 7EP were significant (P <0.05). As shown in the following example, the numerical order of W/R ratios among these three sets is 7EH > 7EM > 7EP, implying that the accessibility of HO 258 to the RBC chromatin is the least among the three DNA dyes tested: W/R ratios of Spl of 7EH, 7EM, and 7EP were 84/48 = 1.75, 76149 = 1.55, and 72/53 = 1.36, respectively.

Coefficients of variation (CVs). CVs of the RBCs were invariably larger than those of WBCs (nearly twice larger). In the MM, 7EP, and 7EH series of Xe- nopus, means of RBC CVs were 11.60 ? 1.61 (n = 24), 11.44 * 1.05 (n = 16), and 15.52 t 2.85 (n = 4) respectively, whereas those of WBC CVs were 5.54 * 1.37 (n = 21),5.97 t 1.49 (n = 161, and 3.3 t 0.687 (n = 5) respectively. These CVs of the RBC peaks were much the same in the different combinations of fixatives and DNA dyes tested, although CVs of microspheres of 9.5 pm used for standardizing instrument performance were routinely maintained at less than, or around, 2%. It is likely, therefore, that the large CVs of RBC peaks resulted, a t least in part, from the flat and elliptic shape of the amphibian RBCs.

50% EtOH Fixation Combined With PI, MI, or H O 258 Staining

Flow cytometric patterns of cell suspensions that were prepared from the same organs as those used in the preceding fixation protocol of 66% MeOH or 70%

FIG. 2. A-C: Cytospin preparations of the cells sorted for Figure 1 B,C,D revealed that cells collected from lower IGFL values of “A windows (AL-AU)” were RBCs, whereas cells from higher IGFL values of “B windows (BL-BU)” were WBCs. AL (BL) and AU (BU) indicate the lower and upper limits of a sorting window of A (B), respectively. D-H RBCs (r) and WBCs (w) of mature Xenopus. D: Living RBCs. Note the flat contour of the cells seen in a lateral view; a protruding middle portion of the cell indicates the site of the nucleus (arrow). E-H: Fixation alters the morphology of RBCs and WBCs. These cells were stained with the fluorescent DNA dyeindicated below, and then cytospun on slides, prior to a double staining with Delafield haema-

toxylin and eosin Y. E: Peripheral blood cells fixed in 70% EtOH, and then stained in modified Vindel0v’s PI without NP40. Note the shrinkage of the cells in comparison with the live cells. F-H: Periph- eral blood cells (F,G) and spleen cells (H) fixed in 50% EtOH, and then stained in modified Vindelerv’s PI without (F) and with (G-H) NP40. Note near disappearance of the RBC’s membrane (indicated by arrowheads) and swelling of the nucleus of the cells, except one which remains seemingly intact (arrow); 50% EtOH-fixed RBCs, including their nucleus, swell considerably more in modified Vindelev’s PI solution with NP40 (G,H) than in the solution without NP40 (F). Bar = 20 Fm.


EtOH, but fixed in 50% EtOH (and then stained with PI, MI or HO 258), differed markedly from those of the cell suspensions that were fixed according to the preceding protocols (Fig, 1, 5EP). Every cell suspension showed an unimodal distribution of DNA contents, as illustrated in the single-parameter histograms (Fig. 1, fifth column). These unimodal peaks were much closer, if not identical, to the peaks of the WBCs in the same specimens that were analyzed at the same instrument gain settings after they were fixed according to the preceding fixation protocol. These unimodal curves, however, were often skewed slightly to the left, and in a few instances? the “unimodal” peak had an extra peak heavily overlapped with the major one.

Based on the foregoing observations, it is presumed that fixation with 50% EtOH causes the chromatin of RBCs to convert conformationally so that it becomes equivalent to that of WBCs. Consequently, the altered chromatin of these RBCs could bind fluorescent DNA dyes stoichiometrically as does the chromatin of WBCs. Under “unfavorable circumstances,” however, the chromatin of some RBCs eluded its alteration to various degrees: samples containing such RBCs with various degrees of alterations thus yielded the histograms that displayed the unimodal curve skewed to the left in varying degrees, or occasionally the heavily overlapped bimodal curves. The following observations on the morphology of hemopoietic cells after they were fixed with 50% EtOH support this assumption.

Under this fixation protocol, the nucleus of both RBCs and WBCs was considerably swollen; the RBC’s nucleus was so enlarged that it was equal to, or even larger than, that of the swollen WBC’s nucleus (Fig. 2F-H). This suggests that this fixation caused “decondensation” of the chromatin in the RBCs. Moreover, the cell membrane of the RBCs lost its rigidity and became optically fuzzy. These changes were reflected by the LSPW peak value which is lower than that of 70% EtOH-fixed specimens, indicating that the sizes of RBCs measured by LSPW in these specimens were mostly those of their fluorescent “nu~leus’~ (see Fig. 3). Although the “decondensation” of chromatin was observed in the majority of RBCs in every sample, there were always a few RBCs which appeared unaffected, retaining their small and compact nucleus and optically reflective intact cell membrane (Fig. 2F).

Modified VindelGv’s PI staining solution that contained NP40 appeared to have very little if any effect on the morphology of RBCs and WBCs that were fixed in 70% EtOH. But when the same staining solution was used on the samples that were fixed by 50% EtOH, it appeared to enhance decondensation of chromatin, in that the nuclei of both RBCs and WBCs in the sample that were stained by this solution were enlarged (Fig. 2F-H) much more than those of the RBCs and WBCs that were stained in the PI solution free of NP40 (Fig. 2F). Moreover, as shown in Figure 2H, the cell membrane of RBCs was much more blurred. However, a few morphologically unaltered RBCs remained after 6 h in

this solution at room temperature. The CVs of unimodal distribution peaks obtained from modified Vindel- ov’s PI staining, regardless of whether it contained NP40, were rather tight, resembling the CVs of the WBC peaks in the samples which were fixed by either 70% EtOH or 66% MeOH. CVs of the samples that were stained by MI (6.61 * 1.79) were similar to those of the samples stained by PI (6.47 t 1.611, whereas the CVs of those stained by H0258 were somewhat larger (8.5 * 0.65).

Analysis of a 1:l Mixture of 2N and 3N Hemopoietic Cells

Hemopoietic cells of 3N are larger than those of 2N, and also fluorescent DNA values of 3N cells are approximately 1.5-fold greater than those of 2N cells as shown in the histograms of Figure 3. Seemingly, therefore, no difficulty exists in distinguishing the cells of 3N from those of 2N by flow cytometry according to their different LSPW (reflecting their sizes) and fluorescence values (reflecting their DNA contents) in a given chimeric sample which contain cells of both ploidy levels. This was indeed the case when the analyses were carried out on the chimeric samples that were fixed in 50% EtOH; however, i t was not the case when 70% EtOH (or 66% MeOH) was used.

Chimeric specimens fixed in 70% EtOH or 66% MeOH. Hemopoietic cells of a 3N adult, fixed in 70% EtOH or 66% MeOH, demonstrated a bimodal DNA distribution representing RBCs and WBCs, respectively, as was the case in 2N hemopoietic cells. Thus, it is clear that if a 2N sample with a bimodal DNA distribution is mixed with a 3N sample which also displays a bimodal DNA distribution, the histograms resulting from such a mixture will be confusing, to say the least, especially in view of the fact that CVs of the RBC peaks in both 2N and 3N are extremely large. This is precisely what happened when 2N and 3N hemopoietic cells were fixed in 70% EtOH or 66% MeOH (regardless of the subsequent staining, MI, PI, or HO 258). In the example shown in Figure 3 (fifth and sixth columns), hemopoietic cells of Kid from a 2N adult were mixed with an equal number of cells from Kid of a 3N adult, fixed in 70% EtOH, and stained with MI. As shown in Figure 3 (2,3NK7EM) neither one- nor two-parameter histograms of this mixed-ploidy sample revealed any clear separation of the four cell groups representing the RBC and WBC of 2N (2NK7EM) and 3N (3NK7EM), except 3N WBC. The Pbl, Spl, and Liv of the similarly mixed 2N and 3N displayed similarly confusing histograms. Analyses of the data from these samples by means of a PDP 11/73 microcomputer (DEC., Maynard, MA), using homebuilt software pack- ages, could not resolve 2N WBCs from the overlapping 3N RBCs or clearly separate 2N RBCs from 3N RBCs. Thus, it is obvious that for the identification of the ploidy of hemopoietic cells in 2N/3N chimeras, the fixation protocol of 70% EtOH or 66% MeOH should be avoided.


Relative DNA Content

FIG. 3. Histograms obtained from samples where equal numbers of hemopoietic cells from a 2N and a 3N adult of Xenopus were mixed, fixed in either 5 0 6 (first to fourth columns) or 70% EtOH (fifth and sixth columns), and then stained with MI. A Histograms obtained from 2N cells fixed in either 50% EtOH (peripheral blood, 2NB5EM and kidney, 2NK5EM) or 70% EtOH (kidney, 2NK7EM). B Histo- grams obtained from 3N cells fixed in either 50% EtOH (peripheral blood, 3NB5EM and kidney, 3NK5EM) or 70% EtOH (kidney, 3NK7EM). C: Histograms obtained from the mixture of 2N and 3N

Chimeric specimens fixed in 50% EtOH. If the same Kid sample of the mixed ploidy as that used in the preceding protocol was fixed in 50% EtOH, the distinction of the 2N and 3N was unmistakably revealed in both one- and two-parameter histograms (2,3NK5EM in Fig. 3). Similarly, the clear distinction of 2N from 3N cells was achieved in the remaining samples of Pbl (2,3NB5EM in Fig. 31, Spl, and Liv of the mixed ploidy, if they were preserved with this protocol. As shown in the two-parameter histograms, “relative cell size” measured by LSPW of these specimens ranged over lower scales than the ranges of 70% EtOH- fixed specimens. This is likely to have resulted from the loss of reflectiveness of the RBC’s membrane caused by this 50% EtOH fixation. In other words, RBC’s sizes measured by LSPW of 50% EtOH-fixed specimens represented actually the sizes of RBC’s nucleus that were stained with fluorescent DNA dyes rather than those of their whole cell body. To conclude,

cells which was fixed in either 50% EtOH (peripheral blood, 2,3NB5EM and kidney, 2,3NK5EM) or 70% EtOH (kidney, 2,3NK7EM). Total number of cells counted in each sample was 50,000. On the ordinate (relative cell size) of the two-parameter histograms, peaks of the microspheres that were used for the calibration of LSPW are indicated: small arrowheads represent the peak of the microspheres with the diameter of 5.08 p m and the large arrowheads represent the peak with the diameter of 9.6 pm.

this fixation protocol is the only one among all the protocols so far tested which provides a reliable and reproducible separation of 2N and 3N adult hemopoietic cells including mature RBCs.

DISCUSSION Fixations of mature Rana and Xenopus hemopoietic

cells in 66% MeOH or 70% EtOH followed by the DNA staining with either MI, PI, or HO 258 preserved the morphology of the hemopoietic cells, but the flow cytometric analysis revealed that these cells invariably yielded two distinctly separate peaks which were represented by RBCs (at lower fluorescence values) and WBCs (at higher fluorescence values); CVs of the RBC curves were much larger than those of the WBC curves. In contrast, clear unimodal curves of DNA distribution with tight CVs appeared when the cells were fixed in 50% EtOH and stained subsequently in the same DNA dyes. Microscopic examinations of these fixed cells re-

684 HOLTFRETERANDCOHEN

vealed that the nucleus of RBCs and WBCs, as well as the whole cell body of RBCs, were enormously swollen, and both cellular and nuclear membranes lost their rigid appearance. Nonetheless, the presence of both RBCs and WBCs in the fixed cell suspensions verifies that the unimodal DNA distribution did not result from the deletion of either RBCs or WBCs.

The WBC peak value of a specimen which was fixed in 70% EtOH (or 66% MeOH) corresponded to the peak value of the unimodal curve obtained for a sample from the same animal and tissue source that was fixed in 50% EtOH, when both samples were analyzed at the same instrument gain settings. Therefore, i t is clear that the way in which the RBC chromatin was preserved in 70% EtOH (or 66% MeOH) was associated with less DNA dye binding than RBC chromatin fixed in 50% EtOH. This reduction of DNA dye uptake by the RBC chromatin was observed with both groove-binding (MI and HO 258) and intercalating (PI) dyes, although the binding reduction of the latter dye was less than that of the former.

We are unaware of any literature reporting that the percent of alcohol used as a fixative affects stoichiomet- ric binding of fluorescent DNA dyes to the nucleus or chromatin. The DNA profiles (with GI, S, and Gz + M phases) from flow cytometric analyses of mammalian cell lines (e.g., HeLa, W1-38, CHO, and mouse ascites) are identical, regardless of whether the cells were a) fixed and stained simultaneously in MI that was dissolved in 25% EtOH (4) or b) stained in PI after 50% MeOH fixation (18) or c) stained with ethidium bro- mide (EB), PI, or MI after 70% EtOH fixation (5,6). One common characteristic of these cells is that they were all active metabolically, implying that their chromatin was not as condensed as the chromatin of mature (nucleated) RBCs or fully mature spermatozoa. It is possi- ble, therefore, that the reduction of DNA dye uptake by frog RBCs fixed in 70% EtOH (or 66% MeOH) results from the compactness of their chromatin, although it is not improbable that the fixation could have further enhanced the condensation of chromatin existing prior to the fixation.

It has been well documented that during the maturation of chicken RBCs, the nuclear chromatin becomes transcriptionally inert (2,171 and undergoes marked condensation (17) in association with augmentation of the RBC specific histone H5 (25). It is also known that the RBC nucleus of mature Xenopus is far less active in RNA synthesis than that of chicken RBCs (21) and that the mature Xenopus RBCs are biosynthetically inert (12,Zl). Accordingly, it is more likely that the fixation with 70% EtOH and 66% MeOH preserves preexisting compactness of RBCs chromatin as it was, rather than that the fixation causes the chromatin to condense. The following would suggest an affirmative answer to the critical question of whether the condensed RBC chromatin binds DNA dyes less than the metabolically active WBC chromatin. Acridine orange binding to the chromatin of maturing chick RBCs, which were fixed

in ethanol acetone and stained on slides, decreases as their maturation and nuclear condensation increases (17). Similar binding reduction of acridine orange and methyl green has been reported for bull spermatozoa, whose heads consist of condensed chromatin (14). Bind- ing reduction of EB-MI has been also reported for mature spermatozoa from humans, Syrian hamsters, and mice (fixed in 96% EtOH), and it has been reported that this reduction of the dye uptake can be successfully restored by digestion of the 96% EtOH-fixed spermatozoa with papain or pronase (28).

Based on the foregoing information, we suggest that 70% EtOH (or 66% MeOH) preserved the compacted chromatin of frog’s mature RBCs. ‘ In contrast, 50% EtOH altered (decondensed) the compact state of the chromatin to the states configurationally equivalent to the chromatin of WBCs. This, in turn, made the chromatin of RBCs and WBCs equally accessible to the fluorescent dyes. Although the mechanism involved in the reduced binding of DNA dyes to the compact chromatin and the putative decondensation of the RBC chromatin in 50% EtOH fixation remains unknown, the following information might be pertinent. When an aqueous solution of “purified DNA” is exposed to increasing con- centrations of EtOH, the DNA sedimentation constant rises abruptly at about 65% and remains high (13). Moreover, electron microscopically distinct forms of DNA can be visualized following the exposure of DNA to lo%, 30%, and 95% EtOH (the higher the concentration of EtOH, the shorter the length, and larger the diameter of DNA) (19).

It has been reported (15) that distribution patterns of DNA content of spermatozoa vary according to the shape of sperm heads: Molluscan spermatozoa with spherical or cylindrical heads produce “asymmetric distributions” laterally extending to higher fluorescence values. The broad distribution of fluorescence values (large CVs) observed on RBCs fixed in 70% EtOH and 66% MeOH may have resulted, therefore, from not only varying chromatin compactness of RBCs which existed at the time of fixation, but also from the flat and elliptic shape of the RBCs. The tight CVs obtained from specimens in 50% EtOH may have resulted from the altered shape of RBCs (physically softened cell membrane in this fixative could no longer retain the flat shape of the RBCs), in addition to the decondensation of chromatin.

Frog RBCs shrank after fixations in either 70% EtOH or 66% MeOH, but the extent of shrinkage differed significantly in these two alcohols: about 43% in 70% EtOH and 26% in 66% MeOH (in reference to the measurement of their long axis). Whether this type of shrinkage is referable to the manner in which the RBC chromatin is preserved in these fixatives remains unknown, but it is interesting to note that in 95% EtOH, DNA molecules undergo 8.6-fold linear compaction, whereas in 95% MeOH, the linear compaction is only

To summarize, 70% EtOH or 66% MeOH (or higher 5.4-fold (10).


percentages of alcohol) should be avoided for flow cytometric analyses of DNA content (or ploidy) of frog mature hemopoietic cells as fixatives of these cells; in- stead 50% EtOH should be used. On the other hand, the results, such as the bimodal distribution of DNA contents separating RBCs from WBCs, and occasional occurrence of a further separation of WBCs into two groups obtained from the fixation with 70% EtOH or 66% MeOH, may provide some information as to the relationship between the state of chromatin preserved with the fixatives and the accessibility of DNA-binding dyes which may reflect their biosynthetic activity of the cells. According to Darzynkewicz, however, the no- tion that “accessibility of nuclear DNA to intercalating dyes is directly proportional to the extent of transcrip- tion is too simplistic and does not apply to all cell sys- tems” (8).

ACKNOWLEDGMENTS We would like to thank Dr. James Leary, Department of

Pathology, University of Rochester, for his indispensable technical advice during the course of these studies and his critical evaluation of this paper. We also would like to thank Mr. Scott R. McLaughlin for his skillful assistance in making sure that the flow cytometry proceeded smoothly. This re- search was supported by USPHS grant HD 07901.

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Fixation-associated quantitative variations of DNA fluorescence observed in flow cytometric analysis of hemopoietic cells from adult diploid frogs