Cell cycle dependence of Hoechst 33342 dye cytotoxicity on sorted living cells

Summary The effect of staining cellular DNA with the bisbenzimidazole dye Hoechst 33342 on the colony forming efficiency of Chinese Hamster Ovary Cells in different cell cycle phases has been studied. Exposures of 90 and 120 min to 5 M Hoechst 33342 provided a considerable loss of clonogenicity depending on the cycle phase at staining procedure. The G2+M cells reveal to be the most sensitive fraction followed by the G1 cells. The highest resistance was found on S-phase cells with a colony forming efficiency exceeding that of the G2+M fraction by a factor of two.     

Computerized measurement of the DNA content, areas, and autoradiographic grains of the same nuclei: demonstration that lightly (3H)thymidine-labeled bone marrow cells are predominantly in G0/G1 and G2

A computerized "flying spot" microdensitometer and scanning stage have been combined to measure the cellular DNA content, nuclear areas, and autoradiographic grain areas of the same cells. The slide positions of the Feulgen-stained, (3H)thymidine-labeled cells are mapped with the computerized stage, and nuclear DNA content and areas are then determined by integral absorbance measurements at 588 nm. Following autoradiograph preparation, the cells are relocated and the areas of the autoradiographic grains over each nucleus are measured at a light wavelength (625 nm) and an optical density setting (greater than 0.10) that do not detect the Feulgen stain. The microcomputer calculates the portion of each nucleus covered with autoradiographic grains (grain area proportion, GAP), and it links the GAP value to the DNA content of each nucleus in the computer file for subsequent sorting and analysis. By using this system in a study of mouse bone marrow cells labeled in vivo with (3H)thymidine, we found that all S-phase cells were clearly labeled after 8 or more days of autoradiographic exposure. Prolonged exposures (up to 64 days) led to detection of lightly labeled cells (0.1 less than GAP less than 0.8) with G1/G0 and G2 DNA content.     

Simultaneous analysis of DNA content and surface antigens in human bone marrow

In order to identify when cellular expansion occurs during hematopoietic maturation, a method was developed for the simultaneous analysis of one or two cell-surface antigens and DNA content on bone marrow cells while preserving their light-scatter properties. Proliferation in a population defined by light-scatter and surface-antigenic characteristics was assessed by measuring the percentage of cells in this population having more than 2C amount of DNA ("proliferation index"). Viable, low-density (1.077 g/cm3), bone marrow cells, stained with monoclonal antibodies conjugated with fluorescein or phycoerythrin, were fixed with paraformaldehyde and subsequently treated with the detergent, Tween 20. The UV-excitable DNA stain Hoechst 33342 was used to quantify DNA content in the cells without interference with immunofluorescence. A FACS IV flow cytometer was used, equipped with the first laser at 488 nm emitting for light scattering and immunofluorescence measurements and the second laser emitting at 360 nm for the Hoechst excitation. The Hoechst uptake was the same for all bone marrow populations, yielding a tight coefficient of variation (CV) (average 5.0%) for the G0/G1 DNA peak. This permitted high sensitivity of cell detection in S, G2, and M phases of the cell cycle, while preserving light-scattering properties of the cells and maintaining cell surface immunofluorescence. The lowest "proliferation index" detected using this technique was 0.08% in a sample obtained from a patient with chronic lymphocytic leukemia. Normal helper T lymphocytes in marrow had approximately 0.5% of the cells in S, G2, or M phase. We show that the erythroid lineage, in the adult normal bone marrow, is the most active in proliferation among all hematopoietic lineages.     

DNA and nuclear protein measurement in columnar epithelial cells of human endometrium

Propidium iodide DNA flow cytometry, Feulgen-DNA, and nuclear light green protein scanning cytometry were performed in columnar epithelial cells of normal, nonmalignant human endometrium and endometrial adenocarcinomas. Columnar cells were identified by immunohistochemical staining for cytokeratin 18, an intermediate filament protein specifically present in columnar cell epithelium. DNA measurements derived from flow and scanning cytometry showed comparable results. The DNA content of the G0/G1 fraction of the adenocarcinomas had a considerable overlap with that of normal endometrium, with that of the carcinomas shifted toward higher values. For the carcinomas, no correlation was found with the histological grade, with the exception of the adenosquamous carcinomas. Most of the clinical stage I tumors showed a DNA content in the normal diploid region. Three of the four carcinomas of clinical stage II and higher had an increased DNA content. For the carcinomas, the percentage of cells in the proliferative fraction, as determined from scanning cytometric derived DNA histograms, was comparable to that of normal endometrium, or higher. No correlation was found with the histological grade. Tumors of clinical stage II and higher had intermediate values compared to carcinomas of lower stages. The nuclear protein/DNA ratio of malignant endometrium completely overlapped that of normal endometrium. Within the tumor population, no correlation was found with the histological grade, with the exception of the adenosquamous carcinomas, and clinical stage. Based on the aforementioned parameters, no discrimination could be obtained between normal and malignant endometrium. However, when the DNA content of the G0/G1 fraction was combined with the coefficient of variation of the nuclear protein/DNA ratio, a clear discrimination could be obtained with only two false-positive cases.     

Detection of cell cycle subcompartments by flow cytometric estimation of DNA-RNA content in combination with dual-color immunofluorescence

BACKGROUND: Correlated flow cytometric measurements of phenotype and DNA-RNA content offer detailed information on cell cycle status of subpopulations in heterogeneous cell preparations in response to stimulation. We have developed a method for flow cytometric analysis of DNA-RNA content that has been optimized for simultaneous measurement of dual-color immunofluorescence. METHODS: Nucleic acid staining was performed at low pH in the presence of saponin. DNA was stained with 7-aminoactinomycin D (7-AAD) and RNA with pyronin Y(G) (PY); both dyes were used at low concentrations, and 7-AAD was exchanged with nonfluorescent actinomycin D after DNA staining to minimize fluorochrome-fluorochrome interactions. For cell surface antigen staining, allophycocyanin was combined with pH-independent Alexa488 instead of fluorescein-isothiocyanate (FITC) because FITC is pH sensitive. RESULTS: This method identified cell cycle subcompartments in CEM cells comparable to published results on cell lines using other dyes and staining methods. Measurement of DNA-RNA content in CD8 lymphocyte subsets of human peripheral blood mononuclear cells costimulated with CD3/CD28.2 showed that, after 48 h of stimulation, 80% of CD8(+) T cells were in the proliferative state, whereas 86% of CD8(+) non-T cells remained in G(0). CONCLUSIONS: This technique permits the clear identification of cellular subpopulations by phenotype and assessment of their cell cycle status.     

Flow cytometric determinations of cellular substances in algae,bacteria, moulds and yeasts

The practical use of flow cytometry is shown in several microbial assays. Recent technical improvements in the optics and electronics of flow cytometric systems as well as in staining techniques permit the measurements of minute cellular components such as the cellular DNA and the protein content of bacteria, algae, moulds and yeasts. Single cell ingredients can be measured by this assay according to their specific stainability. The cell DNA was stained by propidium iodide while the cell protein was fluorochromed by fluorescein-iso-thiocyanate. The DNA synthesis of Saccharomyces cerevisiae and Saccharomyces pastorianus runs discontinuously while the protein content increases continuously during the vegetative growth. The different stages of DNA synthesis of yeast cells can be divided into two gap phases, a synthesis and a mitosis period, corresponding to Howard and Pelc's model of DNA synthesis. Living and dead cells can be counted differentially after staining with Erythrosine B. The red fluorescence of the chlorophyll in algae can readily be used to determine the chlorophyll content of these cells.     

Adaptation of the naphthol yellow S staining for objects with high protein content

Summary In measuring isolated rat liver cells stained with Naphthol Yellow S (NYS) at optimal conditions of pH (2.8), the absorbances measured at the absorption peak of 430 nm appeared to be far too high locally to enable accurate cytophotometric measurements. In order to bring down these absorbances, different techniques for flattening the cells, off-peak measurement and NYS staining at non-optimal pH levels have been applied respectively. Using albumin incorporated in polyacrylamide model films, the reliability of off-peak measurements and the quantitative aspects of the modified protein staining procedures have been investigated. It was found that the NYS procedure can be used as a quantitative protein staining not only at pH 2.8, but also at pH 2.0, 3.5 and 4.0 respectively. The problem with regard to the cytophotometric measuring of isolated liver cells could only be solved, however, by combining a specially developed flattening procedure (by centrifuging small drops of suspension) with staining at non-optimal pH levels. In contrast to the model film results, off-peak measurements applied in situ appeared to give rather unreliable results. In cases of a combined Feulgen-NYS staining, the Feulgen-DNA values were not significantly influenced by any of the modifications of the original NYS staining procedure.     

Adaptation of the Naphthol Yellow S staining for objects with high protein content.

In measuring isolated rat liver cells stained with Naphthol Yellow S (NYS) at optimal conditions of pH (2.8), the absorbances measured at the absorption peak of 430 nm appeared to be far too high locally to enable accurate cytophotometric measurements. In order to bring down these absorbances, different techniques for flattening the cells, off-peak measurement and NYS staining at non-optimal pH levels have been applied respectively. Using albumin incorporated in polyacrylamide model films, the reliability of off-peak measurements and the quantitative aspects of the modified protein staining procedures have been investigated. It was found that the NYS procedure can be used as a quantitative protein staining not only at pH 2.8, but also at pH 2.0, 3.5 and 4.0 respectively. The problem with regard to the cytophotometric measuring of isolated liver cells could only be solved, however, by combining a specially developed flattening procedure (by centrifuging small drops of suspension) with staining at non-optimal pH levels. In contrast to the model film results, off-peak measurements applied in situ appeared to give rather unreliable results. In cases of a combined Feulgen-NYS staining, the Fuelgen-DNA values were not significantly influenced by any of the modifications of the original NYS staining procedure.     

"Liquidless" cell staining by dye diffusion from gels and analysis by laser scanning cytometry: potential application at microgravity conditions in space

BACKGROUND: Conventional staining of cells or tissue sections on microscope slides involves immersing the slides into solutions of dyes then rinsing to remove the unbound dye. There are instances, however, when use of stain solutions is undesirable-e.g., at microgravity conditions in space, where the possibility of accidental spill (many dyes are known carcinogens) introduces health hazard. Likewise, transporting bulk of liquid stains and rinses may be burdensome in certain situations such as field expeditions or combat. METHODS: The "liquidless" staining procedure is proposed in which the dyes are contained in thin strips of hydrated polyacrylamide or gelatin gels that have been presoaked in the stain solutions. Fluorochromes that have affinity to DNA (propidium iodide, PI; 4,6-diamidino-2-phenylindole, DAPI, Hoechst 33342) or to protein (sulforhodamine 101) were used to saturate the gels. The gel strips were placed over the prefixed cells or tissue sections deposited on microscope slides and relatively low (20 g/cm2) pressure was applied to ensure the contact. The cells were also stained by using commercially available mounting media into which DAPI or PI were admixed. Intensity of fluorescence of the PI stained cells was measured by laser scanning cytometry (LSC). RESULTS: Satisfactory cell and tissue staining, with minimal background, was achieved after 10-20 min contact between the cells and gels. Optimal concentrations of the dyes in the solutions used to presoak the gels was found to be 2-4-fold higher than the concentrations used routinely in cytometry. The measurements of intensity of cellular fluorescence by LSC revealed that the staining of DNA was stoichiometric as reflected by the characteristic cellular DNA content frequency histograms with distinct G1, S, and G2/M cell populations and 2:1 ratio of G2/M to G1 peak fluorescence. Individual gels can be saturated with more than a single dye-e.g., to obtain differential DNA and protein staining. Cell staining with DAPI or PI in the gelatin-based mounting media led to high fluorescence background while staining with DAPI in "aqueous" medium was satisfactory. CONCLUSIONS: Relatively fast staining of cells or tissue sections on microscope slides can be achieved by nonconvective dye diffusion using hydrated gels permeated with the dyes, applied to cells at low pressure. The quality of the staining provided by this methodology is comparable to conventional cell staining in dye solutions.     

A new method to discriminate G1, S, G2, M, and G1 postmitotic cells

A new flow cytometric method combining light scattering measurements, detection of bromodeoxyuridine (BrdU) incorporation via fluorescent antibody, and quantitation of cellular DNA content by propidium iodide (PI) allows identification of additional compartments in the cell cycle. Thus, while cell staining with BrdU-antibodies and PI reveals the G1, S, and G2 + M phases of the cell cycle, differences in light scattering allow separation of G2 phase cells from M phase cells and subdivision of G1 phase into two compartments, i.e., G1A representing postmitotic cells which mature to G1B cells ready to initiate DNA synthesis. The method involves fixation of cells in 70% ethanol, extraction of histones with HC1, and thermal denaturation of DNA. This treatment appears to enhance the differences in chromatin structure of cells in the various phases of the cell cycle to the extent that cells could be separated on the basis of the 90 degrees scatter. Mitotic cells show much lower scatter than G2 phase cells, and G1 postmitotic cells (G1A) show lower scatter than G1 cells about to enter the S phase (G1B). Light scattering is correlated with chromatin condensation, as judged by microscopic evaluation of cells sorted on the basis of light scatter. The method has the advantage over the parental BrdU/DNA bivariate analysis in allowing the G2 and M phases of the cell cycle to be separated and the G1 phase to be analyzed in more detail. The method may also allow separation of unlabeled S phase cells from mitotic cells and distinguish between labeled and unlabeled mitotic cells.     

Combined staining procedures for cytophotometry of protein and DNA Feulgen-Naphthol Yellow S and dinitrofluorobenzene-Feulgen

Summary A comparison has been made between dinitrofluorobenzene (DNFB) and Naphthol Yellow S (NYS) as protein stains in combination with the pararosaniline-SO₂ Feulgen procedure. Chicken erythrocytes were used as test cells. Cytophotometric measurements were made using a Zeiss scanning stage cytophotometer coupled to a PDP 11/10 minicomputer using the BICOSCAN program to obtain values for protein per cell, protein per nuclear area and DNA per nucleus. With 5N HCl as the Feulgen hydrolysis agent, DNFB staining, applied before the Feulgen procedure, was found to be unaffected by hydrolysis conditions required to give optimum Feulgen staining and showed only small losses after longer hydrolysis times. On the other hand measurements of NYS staining, of necessity applied after the Feulgen procedure, seem to be susceptible to the duration of Feulgen hydrolysis. This susceptibility is probably due to the interaction of the DNA phosphates with the basic amino acid residues, potential binding sites for NYS. Since the degree of this interaction may be variable, it is argued that NYS binding will measure the available basicity of proteins at the time of staining but no specific protein fraction. DNFB binding is unaffected by DNA-protein interactions and therefore can give a more reliable measure of nuclear protein, particularly in conjunction with Feulgen-DNA measurements.     

Quantitation of cell kinetic responses using simultaneous flow cytometric measurements of DNA and nuclear protein

A rapid procedure was developed for the simultaneous flow cytometric analysis of nuclear protein using fluorescein isothiocyanate, and DNA using propidium iodide in isolated nuclei. The staining procedure did not involve centrifugation and was easily adapted to the staining of human peripheral blood lymphocytes stimulated with phytohemagglutinin, EL4 murine lymphoid tumor cells in suspension culture, and R3327-G rat prostatic adenocarcinoma solid tumor specimens. Histograms of unstimulated and PHA-stimulated HPBL perturbed by actinomycin D, hydroxyurea, 3H-TdR, colcemid, or hydroxyurea + colcemid showed that 1) resting, noncycling G1 (G1Q) cells are distinguished from late G1 (G1AB) cells, 2) early G2 (G2A) cells are distinguished from late G2 (G2B) cells, and 3) mitotic cells are distinguished from G2 cells. Treatment with hydroxyurea resulted in a build-up of cells having high nuclear protein content and 2C DNA content (G1AB), while incubation with 3H-TdR caused an increase in the number of cells with high nuclear protein content and 4C DNA content (G2B). Colcemid-blocked mitotic cells were identified as having low nuclear protein content (lower than G2A nuclei) and 4C DNA content. The nuclear DNA/protein histograms of untreated and colcemid-treated log-phase EL4 cells provided information concerning G1A, G1B, S, G2A, G2B, and M. The method was also used to quantitate the response of androgen-sensitive rat prostatic R3327-G tumors to androgen deprivation following castration. Sample preparation and staining for correlated nuclear DNA/protein measurements takes approximately the same amount of time as for single parameter nuclear DNA measurements.     

Chromatic shifts in the fluorescence emitted by murine thymocytes stained with Hoechst 33342.

BACKGROUND: Many methods in flow cytometry rely on staining DNA with a fluorescent dye to gauge DNA content. From the relative intensity of the fluorescence signature, one can then infer position in cell cycle, amount of DNA (i.e., for sperm selection), or, as in the case of flow karyotyping, to distinguish individual chromosomes. This work examines the staining of murine thymocytes with a common DNA dye, Hoechst 33342, to investigate nonlinearities in the florescence intensity as well as chromatic shifts. METHODS: Murine thymocytes were stained with Hoechst 33342 and measured in a flow cytometer at two fluorescence emission bands. In other measurements, cells were stained at different dye concentrations, and then centrifuged. The supernatant was then used for a second round of staining to test the amount of dye uptake. Finally, to test for resonant energy transfer, we measured fluorescence anisotropy at two different wavelengths. RESULTS: The fluorescence of cells stained with Hoechst 33342 is a nonlinear process that shows an overall decrease in intensity with increased dye uptake, and spectral shift to the red. Along with the spectral shift of the fluorescence to the longer wavelengths, we document decreases in the fluorescence anisotropy that may indicate resonant energy transfer. CONCLUSIONS: At low concentrations, Hoechst 33342 binds to the minor groove of DNA and shows an increase in fluorescence and a blue shift upon binding. At higher concentrations, at which the dye molecules can no longer bind without overlapping, the blue fluorescence decreases and the red fluorescence increases until there is approximately one dye molecule per DNA base pair. The ratio of the blue fluorescence to the red fluorescence is an accurate indicator of the cellular dye concentration.     

Sequential paraformaldehyde and methanol fixation for simultaneous flow cytometric analysis of DNA, cell surface proteins, and intracellular proteins.

A cell fixation and permeabilization procedure consisting of sequential paraformaldehyde and methanol was evaluated and found suitable for concomitant flow cytometric quantification of total cellular DNA, immunofluorescence measurements of cell surface proteins, and immunofluorescence measurements of intracellular proteins. Paraformaldehyde/methanol-fixed cells exhibited significantly greater intracellular antitubulin immunofluorescence than cells fixed with paraformaldehyde or methanol alone (p less than 0.002) and significantly greater intracellular antitubulin immunofluorescence than cells fixed with methanol followed by paraformaldehyde (p less than 0.006). With paraformaldehyde/methanol fixation, cell morphology was well preserved and forward and right angle light scatter properties were sufficiently well maintained to permit gating on these parameters. Cell surface marker staining with fluorescent anti-leukocyte antibodies was unaffected by fixation with paraformaldehyde/methanol. Paraformaldehyde effects on the intensity of DNA staining with propidium iodide were dependent on paraformaldehyde concentration and fixation temperature; these effects were least pronounced at low paraformaldehyde concentrations (0.25% or less), and at temperatures lower than 37 degrees C. Paraformaldehyde fixation may result in differences in propidium iodide staining of DNA in some diploid cells, which may produce small spurious aneuploid peaks in normal peripheral blood leukocytes. Paraformaldehyde fixation also produces an apparent increase in the DNA index of aneuploid cell populations in comparison with methanol fixation, particularly when the DNA index exceeds 1.5. Occasionally, this paraformaldehyde fixation-induced effect is useful in identifying biologically distinct near-diploid subpopulations in tumors.     

Biochemical and morphological identification of ceramide-induced cell cycle arrest and apoptosis in cultured granulosa cells

We have investigated the effects of ceramide on the progression of cell cycle and on apoptotic cell death in ovarian cultured granulosa cells. Rates of cellular proliferation were measured by immunocytochemical staining for proliferating cell nuclear antigen (PCNA) and flow cytometric cell cycle analysis. We also examined for morphological and biochemical signs of apoptosis. The PCNA expression was downregulated in a dose-dependent manner after treatment with C6-ceramide. Flow cytometric analysis demonstrated that the exposure of granulosa cells to C6-ceramide markedly decreased the population associated with G0/G1 DNA content and the reduction of cell numbers in G0/G1 phase was accompanied by the elevation of the A0 phase. The exposure of granulosa cells to exogenous C6-ceramide induced drastic morphological changes including cytoplasmic- or nuclear condensation and typical apoptotic DNA degradation. We also observed that phorbol 12-myristate 13-acetate, a protein kinase C (PKC) activator, significantly inhibited the ceramide-induced apoptosis. These results suggested that ceramide might block the progression of cell cycle at G0/G1 phase and as a consequence, granulosa cells would be committed to apoptosis. Our findings also indicated that down-regulation of the PKC activity might be involved in the ceramide-induced apoptosis in cultured granulosa cells.     

Quantitative analysis of high-resolution trypsin-Giemsa bands on human prometaphase chromosomes

Summary We have constructed ideograms of human prometaphase chromosomes from synchronized and from standard 72-h lymphocyte cultures. G banding was achieved by a trypsin-Giemsa (or Wright's stain) method.In addition to light (white) and dark (black) bands, we have distinguished three different shades of grey. This distinction is essential for proper identification of the increasing number of bands displayed by high-resolution chromosomes. The relative amount of chromatin in each category of staining intensity has been calculated and expressed as light value.The ideograms represent the maximal number of bands discernible with some consistency on prometaphase chromosomes, i.e., 721 euchromatic and 62 variable heterochromatic or heteromorphic bands.The ideograms are based on measurements. On selected printed copies of each chromosome derived from different cells and different individuals, the relative width of each band was measured in relation to the length of the respective chromosome arm. The measurements per chromosome were averaged and used for construction of the ideograms. The distance of each border between bands or sub-bands from the centromere has been calculated on a relative scale, with positions 0 at the centromere and 1.0 at the p terminus or q terminus.The numbering system for bands and sub-bands follows the Paris Conference (1971) recommendations.     

Simultaneous quantification of c-myc oncoprotein, total cellular protein, and DNA content using multiparameter flow cytometry

Variations in total cellular protein content can confound interpretation of the significance of modulations of specific cellular proteins. In an effort to overcome this problem, a technique is described for the simultaneous measurement of a specific cellular protein, total cellular protein, and DNA content. The method utilizes dual-laser (uv and 488 nm) excitation and three fluorescent dyes: FITC, SR101, and DAPI. FITC-labelled antibody coupled with indirect immunofluorescence was used to quantify the c-myc oncoprotein, whereas SR101 and DAPI were used to measure total cellular protein and cellular DNA, respectively. Flow cytometric measurements of c-myc oncoprotein were compared to densitometric readings of p64c-myc. SR101 protein determinations were compared to those obtained by the Lowry technique. Results indicated that flow cytometric measurements correlated well with those obtained by the biochemical methods. The usefulness of the technique was further examined following treatment of exponentially growing HL-60 cells with 2.5 micrograms/ml cycloheximide for 0 to 12 h. Cycloheximide treatment was found to cause a significant decrease in c-myc oncoprotein content within 2 h (P less than 0.05), a relative increase in the proportion of G0/G1 cells and a modest decrease in total cellular protein. This technique appears to provide a rapid, quantitative approach, useful for investigating alterations in cellular growth balance occurring with cell differentiation, neoplastic transformation, or cell treatment with radiation or cytostatic drugs.     

Demonstration of the cell cycle positions of taxol-induced "asters" and "bundles" by sequential measurements of tubulin immunofluorescence, DNA content, and autoradiographic labeling of taxol-sensitive and -resistant cells

We used reliable and relatively inexpensive equipment to make sequential sets of measurements of antitubulin immunofluorescence, Feulgen staining, and autoradiography on the same cells. This was done to evaluate tubulin conformations, DNA content, and [3H]-thymidine incorporation in cell lines sensitive (HL60) and resistant (K562) to the novel anti-tubulin chemotherapeutic agent taxol. Numbers of cells with microtubule bundles have been found to correlate with sensitivity to taxol by clonogenic assay for several leukemic cell lines. We have found that cells with "asters" produced by taxol exposure are in mitosis and that cells with taxol-induced "bundles" are in G0/G1, S, and G2 phases. We further found that S-phase cells with microtubule bundles in both sensitive (HL60) and resistant (K562) cell lines were able to incorporate [3H]-thymidine after 4-hr exposure to taxol. As microtubule bundles and asters occur in cells of the same cell cycle phases in both lines, we conclude that the greater frequency of cells with microtubule bundles reported for sensitive cells after taxol treatment cannot result from drug exclusion nor from different effects of the drug on cell microtubules in these two leukemic lines.     

Cytofluorometric analysis of cell proliferation and differentiation of the human erythroblasts

Summary The method of cytofluorometric measurement of the contents of Hb and nuclear DNA on a single erythroid cell (Fukuda et al., 1975, 1977 a) was used for the quantitative analysis of the erythropoiesis in normal human bone marrow.The intracellular Hb in an erythroid cell was converted to fluorescent porphyrin after removing the Giemsa staining by irradiation with violet light in the presence of SH-donor (mercaptoethylamine hydrochloride, MEA) and its nuclear DNA was subsequently stained with pararosaniline Feulgen staining. With the two quantitative parameters, Hb content and DNA amount, the erythroid cells in normal human bone narrow were classified into 6 classes of different maturation stages (EI-EIV).The morphological characteristics of the most primitive erythroblast (EI cells) were described. The proerythroblasts which were identified on the bases of morphological criteria had in general aneuploid amounts of nuclear DNA with disproportional contents of Hb, thereby indicating that they are rather aberrations from the normal steps of cell maturation. The DNA amounts of orthochromatic erythroblasts (EV cells) showed continuous decrease from diploid range to almost zero suggesting that the removal of nuclear DNA from the erythroblast is not exclusively due to mechanical expulsion of a whole intact nucleus.Partly supported by Alexander von Humboldt-Stiftung and Deutsche Forschungsgemeinschaft DFG-Grant Bo 395/3     

Flow cytometric analysis of unbalanced control of protein accumulation and DNA synthesis in differentiating mouse myeloid leukemia cells

The protein and DNA contents of mouse myeloid leukemia M1 (clone B24) cells were determined by flow cytometry (FCM) after double fluorescent staining of the cells with fluorescein isothiocyanate and propidium iodide. FCM analysis showed that there was a linear relationship between the DNA and protein contents in logarithmically growing cells, although the protein content showed some variation. B24 cells can be induced to differentiate into macrophage-like cells by treatment with a protein inducer(s) in conditioned medium (CM) of hamster embryo cells. When the cells were treated with various concentrations of CM, cells with a 2C DNA content, G1/0 cells, increased and protein accumulated in these G1/0 cells. The increases in the number of G1/0 cells and in their protein content per cell were proportional to the concentration of CM. Serial analysis of changes in the contents of DNA and protein in differentiating B24 cells showed that DNA synthesis was suppressed by differentiation-induced block of the cell cycle at the G1/0 phase, whereas increase in the protein content was not completely suppressed by block of the cell cycle. These results suggest that unbalanced control of the DNA and protein contents of B24 cells is involved in the mechanisms of the morphological changes during differentiation into macrophages.