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A method described to purify pluripotent hemopoietic stem cells ( PHSC ) from adult mouse bone marrow. The method consists of three separation steps. First, bone marrow cells are centrifuged in a discontinuous metrizamide gradient and simultaneously labeled with wheat germ agglutinin-fluorescein isothiocyanate (WGA-FITC). Second, the low density cells are analyzed by a fluorescence-activated cell sorter (FACS) and the WGA-positive cells with medium forward and low perpendicular light scatter intensities are sorted. The WGA-FITC is removed from the cells by incubation with N-acetyl-D-glucosamine. Finally, the sorted cells are incubated with anti-H-2K-biotin and avidin-FITC and sorted a second time to enrich cells with high H-2K density. The sorted cells gave rise to 2 spleen colonies per 100 injected cells at 8 d and 6.6 colonies per 100 cells at 12 d after transplantation into lethally irradiated syngeneic recipients. The average enrichment factor for day 12 CFU-S (colony-forming unit/spleen) was 135 (range, 90--230; n = 15) and was similar to that for the cell type that provides radioprotection (180 +/- 70), indicating that these functional properties were copurified. Indirect evidence suggests that the spleen-seeding efficiency (f factor) of these cells is 0.10 and, therefore, the average purity of the sorted PHSC was 65% (range in 15 experiments, 35--110%). The sorted cells were all in the G1 or G0 phase of the cell cycle. They appeared to be undifferentiated blasts by morphological criteria. Electron microscopy revealed that the sorted cells consisted primarily of two cell types, possibly representing G0 and G1 cells. The FACS was used to deposit single selected cells into individual microwells of Terasaki trays. 32% of the sorted cells could be induced to form myeloid progeny in vitro. This procedure should be useful for direct studies on the regulation of hemopoietic cell differentiation.

A method using flow cytometry and fluorescent in situ hybridization (ISH) to detect RNA in cells is described. Ll210 murine leukemia cells were fixed with 1% formaldehyde in HEPES buffered Hank's balanced salt solution (HH) followed by 70% ethanol. Endogenous RNAses were blocked by diethylpyrocarbonate treatment. Single-stranded sense and antisense RNA probes, labeled with biotin-11-UTP, were transcribed from a 2.1 kb 28s ribosomal RNA (rRNA) gene fragment subcloned into the pGEMZ plasmid. For good results, it was essential that the probes were degraded to 100-150 nucleotides before use. Hybridization was performed at 45°C in 50% fomamide, 5 x SSC, 0.5% SDS. Hybrids were detected with streptavidin-FITC by flow cytometry. Antisense rRNA probe signal was Several in situ hybridization (ISH) methods have been described that allow for the detection of specific nuclei acid sequences with nonradioactively labeled probes. Probes have been labeled directly with fluorochromes (3) or cytochemically detectable enzymes (29). Post hybridization immunocytochemical development steps are not needed for such directly labeled probes. Indirect methods employ probes that are labeled by a hapten or affinity label, which is detected by (immuno-) cytochemical means subsequent to the hybridization reaction. Examples are the use of biotinylated probes (15,22,23), probes labeled with N-Acetoxy-N-Acetylaminofluorene (AAF) (2 1,33), or mercurated probes, which are haptenized after the hybridization reaction with a sulfhydryl-TPN (5,17). Probe labeling is not needed when antibodies specific for RNA.DNA hybrids are used to detect hybrids in situ by immunofluorescence (27,28,31).The availability of a fluorescent ISH method would allow its application to flow cytometry (2). The advantages of such flow cytometric ISH would be the simple quantification of hybridization signals and the rapid analysis of many cells, so that heterogeneous populations or aberrant expression levels in small subpopulations of cells can be analyzed. However, hybridization 100 times higher than the background. The hybrids were largely resistant to RNAse and melted at high temperature, The sense probe also gave a signal (5 times background), which was not RNAse resistant and was attributed to the presence of internal inverted repeats in the ribosomal RNA. When sufficient background reduction can be achieved, it is expected that as few as ten mRNA molecules per cell can be detected with the fluorescent in situ hybridization method.Key terms: Suspension hybridizaton, RNA detection, ribosomal RNA, Ll210 cells, biotinylated probes, single-stranded RNA probes, antisense RNA, nonisotopic nucleic acid probes conditions may seem incompatible with keeping cells, chromosomes, or nuclei in suspension, as is required for flow cytometry. Several papers have been published describing the use of fluorescent ISH to detect DNA targets in isolated interphase nuclei or chromosomes in suspension (12,26,34,35).To our knowledge the present paper is the first to describe the application of fluo...

A fluorescent in situ hybridization procedure with a chromosome 1-specific (lq12) repetitive satellite DNA probe was used to label the lq12 regions of the chromosomes 1 in spherical and polymorphic hemopoietic cell nuclei. The entire procedure was performed in suspension to preserve nuclear morphology. The result was studied by three-dimensional analysis, as provided by a scanning laser confocal microscope. The lq12 regions of chromosome 1 were measured to be closely associated with the nuclear envelope in isolated nuclei of unstimulated diploid human lymphocytes. The relative positions to each other in the periphery of these spherical nuclei could not be distinguished from a random distribution pattern. In the diploid and tetraploid polymorphic nuclei of cells of the promyelocytic leukemia cell line HL60 these pericentromeric sequences were also associated with the nuclear surface.Key terms: Chromosome topography, 3-D image analysis, Kolmogorov-Smirnov test, lq12 probe, biotin-avidin, blood cells In 1885, Rabl (34) proposed a model of intranuclear chromosome topography that is still under investigation. He assumed that the alignment of centromeres and telomeres, present in the mitotic cell, is preserved throughout cell cycle ("Rabl orientation"). A few years later (61, Boveri showed that, in Ascaris eggs, chromosomes occupied a discrete territory in the interphase nucleus, the chromosome "domain." Almost a century later, the understanding of the intranuclear architecture of chromosomes has not increased much despite various efforts (for reviews on this subject, see refs. 9,19). A Rabl orientation was seen in mammalian cells (11) and in Drosophila (14,27).An important technique with respect to the study of the spatial organization of chromosomes within nuclei was introduced by Agard and Sedat (1). They applied optical sectioning of intact cells by sampling images of successive planes, while moving the microscope focus stepwise through the DAPI-stained nucleus. After using deconvolution algorithms to eliminate the out-offocus fluorescence, three-dimensional reconstructions were made to analyze the DAPI banding patterns of polytene chromosomes in intact Drosophila nuclei. The advent of confocal microscopes greatly facilitated 3-D reconstruction and analysis of cells, since deconvolution of the images was not longer required (7,46).The introduction of non-isotopic in situ hybridization (ISH) enabled investigators to tag specific nucleic acid sequences with high speed and high visual resolution on chromosomes and in cell nuclei with a fluorescent or enzymatic label (3,18,21,22,29,32,40). It appeared to be very suitable to detect numerical chromosome aberrations in interphase nuclei, using repetitive satellite DNA probes (10,471. These probes hybridize to the (peri)centromeric heterochromatin on specific chromosomes. Recently, several investigators reported the detection of target sequences on metaphase chromosomes as small as 1 to 5 kb (2,15,23), indicating the high sensitivity achievable with non-isotopic ISH.'Thi...

Conflicting results have been published on whether or not myelodysplastic syndromes (MDS) affect all cell lineages. Involvement of myeloid and erythroid cell lineages has been regularly observed, but it remains controversial whether the different lymphoid cell lineages are involved. In this study of eight patients with MDS associated with monosomy 7, fluorescent in situ hybridization (FISH) was used to enumerate the chromosomes 7 in interphase cells. With the probe D7Z1, the rate of false-positive detection of monosomy 7 was 3% +/- 2% in normal cells. T- and B-cell lines were established from eight patients with MDS and monosomy 7. As determined by FISH in interphase cells, 1.9% (0% to 3%) of the cells in the B-cell lines showed one fluorescent spot and 1.1% (0% to 2.9%) of the cells in the T-cell lines. These values do not differ from normal values. However, the possibility that normal cells were selected when the T- and B-cell lines were established could not be excluded. Therefore, peripheral blood cells were obtained, separated according to surface markers specific for lymphoid and myeloid cell lineage with a cell sorter, and analyzed for the expression of monosomy 7 by FISH. Antibodies recognizing T cells (CD3), B cells (CD20), natural killer (NK) cells (CD57), monocytes and granulocytes (low and high expression of CD11b antigen), and myeloid progenitors (CD33) were used to separate cells. The expression of monosomy 7 in the T cells, NK cells, and B cells did not differ from control values. These results in the lymphoid subpopulations are in stark contrast with the observations in the myeloid populations; the percentage of cells with monosomy 7 ranged from 9% to 78% (controls: 6% +/- 2%) in cells with low CD11b expression, 20% to 89% in cells with a high expression of the CD11b antigen (controls: 7% +/- 3%), and 23% to 91% in the CD33 positive cells (controls: 5% +/- 3%). The results of this study suggest that monosomy 7 does not usually affect lymphoid subpopulations but is restricted to committed progenitor cells with the capacity to differentiate into mature myeloid cells.