Regional differences in the compaction of chromatin in human G0/G1 interphase nuclei.

The large-scale structure of chromatin corresponding to G- and R-bands in human G0/G1 interphase nuclei was compared. Fluorescence in situ hybridization (FISH) was used to measure the interphase distance between 42 pairs of probes separated by 0.1-1.5 Mbp. The probe pairs were derived from 21q22.2 and Xp21.3, G-band positive regions, and from 4p16.3, 6p21.3, and Xq28, R-band positive regions. Distributions of measured interphase distances in all regions approximated a Rayleigh distribution, suggesting that the chromatin follows a random-walk path over this range. A linear correlation of mean-square interphase distance and genomic separation, also indicative of random-walk folding, was observed in all regions. The slope of the correlation observed using probes from G-band regions was systematically lower than that from R-band regions. The difference in the slope between Xp21.3 and Xq28 was particularly striking and was observed in normal fibroblast cells, fixed alternatively with methanol and acetic acid or paraformaldehyde, and HeLa cells. These results demonstrate regional differences in large-scale chromosome structure during interphase, with the more openly configured chromatin corresponding to R-bands.

Optical plankton analyser: a flow cytometer for plankton analysis, II: Specifications.

An analysing flow cytometer, the optical plankton analyser (OPA), is presented. The instrument is designed for phytoplankton analysis, having a sensitivity comparable with commercially available flow cytometers, but a significantly extended particle size range. Particles of 500 microns in width and over 1,000 microns in length can be analysed. Sample flow rates of up to 55 microliters/s can be used. Also, the dynamic range of the instrument is significantly increased for particles larger than about 5 microns. The optics, hydraulics, and electronics of the instrument are described, including the best form for a low fluid shear cuvette. The new pulse quantification technique we call digital integration is presented. This technique is essential for the instrument to handle both short and very long particles with a large dynamic range. Test measurements demonstrating particle size range and dynamic range are presented. Dynamic ranges of 10,000 and 100,000 were typically observed, measuring field samples with Microcystis aeruginosa colonies, whereas one sample showed a dynamic range of 10(6). A simple method for interpretation of time of flight (TOF) data in terms of particle morphology is presented. The specifications of the instrument are given.

High-speed chromosome sorting.

Dual-beam high-speed sorting has been developed to facilitate purification of chromosomes based on DNA staining with the fluorescent dyes Hoechst 33258 and chromomycin A3. Approximately 200 chromosomes per second of two types can be sorted from a suspension of chromosomes isolated from human lymphoblasts while fluorescent objects (chromosomes, debris fragments, chromosome clumps, and nuclei) are processed at the rate of about 20,000 per second. This sorting rate is approximately ten times that possible with conventional sorters. Chromosomes of a single type can be sorted with a purity of about 90 percent. DNA from the sorted chromosomes is suitable for construction of recombinant DNA libraries and for gene mapping.

The binding kinetics and interaction of DNA fluorochromes used in the analysis of nuclei and chromosomes by flow cytometry.

The interactions and binding characteristics of DNA dyes used in the flow cytometric analysis of chromatin were studied using human chromosomes and mouse thymocyte nuclei. The kinetics of dye binding and the relationship between fluorescence intensity and dye concentration are presented. Under the conditions used, Hoechst 33258, propidium iodide and chromomycin A3 reach an equilibrium with thymocyte nuclei after approximately 5 min, 20 min and more than 1 h, respectively. The same binding kinetics are observed with Hoechst 33258 and chromomycin when nuclei are stained with a mixture of the two dyes. Sodium citrate, which improves the resolution of flow karyotypes, causes a rapid increase in Hoechst and propidium iodide fluorescence, but a decrease in the fluorescence of chromomycin. The relative peak positions of chromosomes in a flow karyotype are unaffected by sodium citrate addition. The spectral interaction between Hoechst and chromomycin is quantified. There is variation among the human chromosome types in the amount of energy transferred from Hoechst to chromomycin. By measuring the Hoechst and chromomycin fluorescence of each chromosome after Hoechst excitation, it is shown that the amount of energy transferred is correlated to the ratio of the amount of Hoechst to chromomycin bound. Although the energy transfer between the two dyes is considerable, this has little effect on the reproducibility of flow karyotype measurements. The relative peak positions of all human chromosomes in a 64 X 64 channel flow karyotype, except for the 13 and Y chromosomes, vary in the order of 0.5 channel over a 16-fold change in either Hoechst or chromomycin concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of surface antigen labeling on spleen colony formation: comparison of the indirect immunofluorescence and the biotin-avidin methods.

Indirect immunofluorescence techniques for labeling cell surface antigens on murine pluripotent hemopoietic stem cells often result in a reduction of CFU-S numbers. This phenomenon was investigated by comparing indirect immunofluorescence and biotin-avidin methods using anti-T200 and anti-H-2Kk monoclonal antibodies. Mouse bone marrow cells treated with these monoclonal antibodies, alone or in combination with fluorescein conjugates of rabbit antirat or goat antimouse immunoglobulins, respectively, showed reduced numbers of CFU-S. The reduction in CFU-S numbers by anti-H-2Kk antibodies was dependent on the concentration of antibody and on the antigen density on the cells. Near complete CFU-S recovery was obtained with biotin-labeled antibodies and avidin-fluorescein isothiocyanate. The CFU-S recovery obtained was higher with higher numbers of biotin moieties per antibody molecule. Biotinylation itself did not influence the antibody binding properties. The protective effect was independent of the avidin-FITC concentration. Injection of carrageenan, an agent known to block macrophage activity, prevents the reduction of CFU-S recovery caused by anti-H-2Kk antibody treatment. The biotin-avidin procedure permits the measurement of antigen density on pluripotent stem cells through flow cytometry and sorting and full recovery of CFU-S in the in vivo assay.

Thymus regeneration by bone marrow cell suspensions differing in the potential to form early and late spleen colonies.

We have determined the thymus-repopulating capacity of purified hemopoietic stem cells, bone marrow cells from mice injected four days previously with 5-fluorouracil (5-FUBM), and bone marrow cells cultured in the presence of stem-cell-activating factor (SAF; SAFBM). SAF is identical to interleukin 3 (IL-3). Purified stem cells are more enriched in day-12 CFU-S than in day-8 CFU-S. 5-FUBM consists of CFU-S that give rise to late (day-12) spleen colonies. SAFBM contains predominantly CFU-S that give rise to early spleen colonies (days 6-8). There is also a net increase in the number of spleen colony-forming units (CFU-S) in these cultures. Thymus regeneration after transplantation with either purified stem cells or 5-FUBM was delayed approximately three days as compared with that after transplantation with normal bone marrow cells. This delay can be ascribed to the absence of prothymocytes in these preparations. Thymus regeneration by SAFBM was delayed approximately ten days as compared with that after transplantation with normal bone marrow cells. The most likely explanation of these results is as follows. The prothymocytes in normal bone marrow produce a relatively limited offspring in the thymus soon after transplantation. This is rapidly replaced by the offspring of newly formed prothymocytes, the results of differentiation of the pluripotent stem cells. These stem cells also give rise to late spleen colonies. Stem cells that give rise to early spleen colonies appear to have lost the capacity for differentiation into the T-cell lineage.

Preparation and bivariate analysis of suspensions of human chromosomes.

Chromosomes were isolated from a variety of human cell types using a HEPES-buffered hypotonic solution (pH 8.0) containing KCl, MgSO4, dithioerythritol, and RNase. The chromosomes isolated by this procedure could be stained with a variety of fluorescent stains including propidium iodide, chromomycin A3, and Hoechst 33258. Addition of sodium citrate to the stained chromosomes was found to improve the total fluorescence resolution. High-quality bivariate Hoechst vs. chromomycin fluorescence distributions were obtained for chromosomes isolated from a human fibroblast cell strain, a human colon carcinoma cell line, and human peripheral blood lymphocyte cultures. Good flow karyotypes were also obtained from primary amniotic cell cultures. The Hoechst vs. chromomycin flow karyotypes of a given cell line, made at different times and at dye concentrations varying over fourfold ranges, show little variation in the relative peak positions of the chromosomes. The size of the DNA in chromosomes isolated using this procedure ranges from 20 to over 50 kilobases. The described isolation procedure is simple, it yields high-quality flow karyotypes, and it can be used to prepare chromosomes from clinical samples.

Separation of spleen colony-forming units and prothymocytes by use of a monoclonal antibody detecting an H-2K determinant.

The density of H-2K antigens was determined on both the mouse hemopoietic stem cell, using an assay for spleen colony-forming units (CFU-S), and the prothymocyte, using a thymus repopulation assay. This was done by light-activated cell sorting of bone marrow cells labeled first with a biotinylated antibody against H-2Kk and then with avidin-fluorescein isothiocyanate. Almost all CFU-S were found to be present among the 4% bone marrow cells with high forward light scatter (FLS), low perpendicular light scatter (PLS), and bright immunofluorescence. Thymus regeneration by this brightly fluorescent fraction was delayed 3 days compared to thymus regeneration by unsorted cells, although the same number of CFU-S was present in each cell suspension. This delay indicates that differentiation from CFU-S to prothymocytes takes 3 days. The fraction of cells in the FLS/PLS window with dull anti-H-2Kk fluorescence contained few CFU-S and gave rise to a transient thymus regeneration. These findings indicate that the prothymocyte carries fewer H-2K antigens than does the CFU-S. The H-2K antigen is a marker with which CFU-S and prothymocytes can be separated. Therefore, during early T-cell differentiation, the number of H-2K molecules on the cell surface decreases (CFU-S----prothymocyte----cortical thymocyte). During maturation of T cells, a reexpression of H-2K molecules occurs, since lymph node cells and spleen cells were shown to be brightly positive for H-2K antigen.

Analysis of phytoplankton by flow cytometry.

Optical properties of eight algae species were measured on a flow cytometer. Forward and perpendicular light scatter measurements provide information on the size and shape of algae cells. The intensity of chlorophyll fluorescence varies greatly among the studied algae species and can be used to distinguish them. Measurements of chlorophyll fluorescence after excitation with different wavelengths provide a fluorescence excitation spectrum for each species over the available wavelength range. These spectra reflect the different photosynthetic pigment contents of the species. Staining algae cells with the DNA stains, Hoechst 33342 and DAPI, provides two additional optical parameters to distinguish algae populations: blue nuclear fluorescence and yellow granular fluorescence. The combination of these optical measurements enables the distinction of each algae species into a small cluster in a hyperspace of parameters. The automation of phytoplankton analysis on the flow cytometer may lead to the rapid and objective assessment of water quality.

The fluorescence intensity of propidium iodide bound to DNA depends on the concentration of sodium chloride.

While performing DNA analysis with propidium iodide using the FACS-II cell sorter, it was noted that the fluorescence intensity was not constant during the measurements. When different sheath and sample fluids were used, the fluorescence intensity of a given cell population increased or decreased during the measurement, depending on the concentration of sodium chloride in the fluids. By selecting the appropriate sheath and sample fluid combination, the drift in fluorescence intensity can be avoided.

Light scattering properties of murine hemopoietic cells.

The light scatter of cells contains information about cell structure and size. Much of this information can be obtained by measuring the light scattered in a flow cytometer in two directions: forward (1.5 degrees-13 degrees) and perpendicular (65 degrees-115 degrees) with respect to the direction of the laser light. For different mouse bone marrow cell types and Sephadex G-25 beads of 10-50 micron diameter, the forward light scatter intensity can be shown to be linearly proportional to the cross-sectional area. The perpendicular light scatter intensity can be shown to depend both on size and degree of structuredness. Therefore, light scatter measurements may be used to obtain overall morphological descriptions of rare cells. By sorting on the basis of light scatter measurements and by subsequent in vivo and in vitro culture assays, it can be shown that the pluripotent hemopoietic stem cell and three committed progenitor cells which represent consecutive stages in the granulocyte/monocyte differentiation series have diameters of 7.1-7.5 micron, and show a complexity of structuredness which increases with differentiation. Since these cells have a low incidence and are only described by their function, such morphological information cannot be obtained by direct microscopic examination of bone marrow. Furthermore, most measurements by flow cytometers can be improved by simultaneous light scatter measurements. Examples are presented which illustrate this in studies of immunofluorescence, leukemic bone marrow, and stem cell purification.

Structural identity of the pluripotential hemopoietic stem cell.

A review is presented of the experiments that resulted in the identification of a specific morphologic entity representing the pluripotential hemopoietic stem cell (HSC) in mouse bone marrow. This entity was subsequently discovered in concentrated HSC preparations from bone marrow of rats, monkeys, and humans. In the mouse, a set of physical parameters (of the HSC) has been collected which agree with its morphologic description. It was also shown that these physical properties, and a number of cell surface properties, do not enable a distinction between HSC and its immediate descendants, the G/M CFU 1 and the E-BFU. The factors that stimulate proliferation of these three cell types have been isolated from human leukocyte conditioned medium and mouse spleen conditioned medium and were partly purified and characterized. The information at present indicates that the three cell types respond to closely related, if not identical, factors. Direct counts of HSC in electron microscopic preparations of density gradient fractions of different enrichment have been compared with HSC values computed from spleen colony counts and f factors for rat and mouse marrow. A high degree of correlation was found between the two types of observations. The slopes of the regression lines for mouse marrow fractions, for concentrates of normal rat marrow, and for concentrates of cycling rat marrow were the same, namely, 0.5. The deviation of this value from the expected value of 1.0 is probably not due to the use of erroneous f values. It is proposed that the observed discrepancy may be due to heterogeneity of spleen colony forming cells, in that a proportion of them may not be pluripotential.

Separation of subpopulations of in vitro colony forming cells from mouse marrow by equilibrium density centrifugation.

Equilibrium density centrifugation was used to characterise and separate subpopulations of mouse haemopoietic progenitor cells capable of producing colonies of granulocytes and macrophages in vitro. The material used to induce colony formation (CSF) was prepared from an extract of pregnant mouse uteri. This CSF preparation was found to be free of factors modifying the response. Under these culture conditions, in vitro colony forming cells (CFU-c) were found to be relatively homogeneous in their buoyant density. This homogeneity was independent of CSF concentration. A heterogeneous density profile of CFU-c was obtained when various cell fractions were cultured in the presence of CSF and rat blood lysate. The majority of the additional cells which responded to erythrocyte lysate were dense (modal density 1.080 g/cm3) compared to CFU-c which responded to CSF alone (modal density 1.074 g/cm3). It is concluded that in vitro colonies induced by CSF and in vitro colonies grown in the presence of CSF and erythrocyte lysate reflect two different populations of CFU-c.

Attempts to transform murine hemopoietic cells by Rauscher leukemia virus.

Primary Rauscher leukemia virus (RLV)-induced myeloid leukemias can produce many small clones in agar in the absence of a factor needed for the proliferation of normal myeloid cells. It seems that leukemic cells can more efficiently utilize the small amount of colony-stimulating factor (CSF) that is produced by them. At optimal stimulation by exogenous CSF, leukemic cells exhibit a poorer rate of proliferation than normal bone marrow cells. Hemopoietic cells can replicate the virus after infection in vitro. In some experiments, infection of normal bone marrow cells leads to the production of some cells with the same growth pattern as cells from primary leukemias.

Antigenic differences between hemopoietic stem cells and myeloid progenitors.

Bone marrow contains pluripotent stem cells which give rise to colonies when injected into irradiated syngenic hosts as well as more differentiated progenitor cells of the myeloid cell which are able to form colonies in vitro. Antisera against brain is known to contain antistem cell antibody. The present experiments were designed to determine if the myeloid progenitor cell still expresses the stem cell antigen. Bone marrow cells were treated with antibrain antiserum plus complement and then survival of stem cells was determined by injection into irradiated hosts. Survival of myeloid progenitor cells was determined by culturing the cells in vitro. It was found that stem cells were eliminated by the antiserum but that myeloid progenitors were not. Inefficient in vitro lysis was ruled out as the reason for this difference since in vitro colonies were not reduced when the cells were treated with anti-immunoglobulin or after passage through an irradiated host. In the differentiation from stem cell to myeloid progenitor there is an associated surface antigen change.