Isolation of murine pluripotent hemopoietic stem cells.

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.

Detection of host cells following sex-mismatched bone marrow transplantation by fluorescent in situ hybridization with a Y-chromosome specific probe.

Fluorescent in situ hybridization (FISH) with a biotinylated Y-chromosome specific repetitive DNA probe was applied to detect Y-bearing cells in blood and bone marrow samples from patients with hemopoietic malignancies after a sex-mismatched bone marrow transplantation. The sensitivity of this method is in the order of 0.1% Y-bearing nuclei in male recipients transplanted with female marrow. In female recipients of male marrow, the detection of low numbers of non-Y-bearing nuclei is less sensitive. The presence of host cells in blood and bone marrow of seven patients (four males, three females) was investigated with respect to successful engraftment or recurrence of the disease. The results obtained were compared with cytology (all seven cases) and with conventional cytogenetics (five cases). In five patients, the results of Y-FISH and cytology were identical. In two patients, low numbers of male host cells were detected in the marrow by Y-FISH, whereas cytology indicated complete remission of the disease. In three patients Y-FISH and cytogenetic data were similar, but in two patients Y-FISH revealed the presence of 0.2% and 7% male host cells, respectively, in bone marrow, whereas cytogenetics indicated a 100% female marrow in both cases. Because the hybridization was performed in situ, the morphology of the nuclei was preserved. To differentiate between normal and leukemic cells, the size of the blast cell nuclei appeared to be a very useful indicator. Our data suggest that fluorescent in situ hybridization with a Y-chromosome specific probe is a fast and sensitive technique to identify the host cells after sex-mismatched bone marrow transplantation, in particular in case of male recipient and female donor combinations.

Light scatter and cell surface properties of murine megakaryocyte progenitor cells.

The characteristics with respect to light scatter and cell surface properties of megakaryocyte colony-forming cells (CFU-Meg), growing in serum-free agar cultures, have been determined. Mouse bone marrow cells were fractionated using a light-activated cell sorter (FACS) either on the basis of light scatter or after staining with a fluorescein isothiocyanate (FITC)-labeled lectin or monoclonal antibody. After staining and sorting, the recovery of CFU-Meg was about 50%. In the unsorted controls 29 +/- 2 CFU-Meg/10(5) cells were observed. The forward (FLS) and perpendicular (PLS) light scatter intensities of CFU-Meg were very similar to those of spleen colony-forming cells (CFU-S) and early progenitor cells. The average diameter of the CFU-Meg as determined by the FLS intensity profile was about 7.5 microns. Wheat germ agglutinin binding of CFU-Meg was high, indicating a high sialic acid content of the cell surface. Expression of the differentiation antigen Pgp-1 on the CFU-Meg as determined by indirect immunofluorescence with the rat monoclonal antibody I42/5.1 was moderate. Pgp-1 expression was lower than the Pgp-1 expression of granulocyte/macrophage progenitors (CFU-C) or granulocytes. It is concluded that the light scatter and cell surface properties of CFU-Meg are similar to those of CFU-S and early committed progenitors.

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.

The use of E. coli exonuclease III to generate single stranded DNA in BrdUrd cell-cycle analysis permits simultaneous detection of cell surface antigens.

An immunocytochemical method for the simultaneous flow cytometric quantitation of total cellular DNA, incorporated 5-bromo-2'-deoxyuridine (BrdUrd) and one or more cell surface antigens has been developed. Biotin labeling of cell surface antigens, critically tuned fixation techniques and an enzymatic denaturation of cellular DNA are the essential features of this method. Enzymatic denaturation of cellular DNA was shown to prevent loss of cell surface antigen-bound biotin moieties, and thus to preserve cell surface immunofluorescence distribution. After a mild protein extraction and the introduction of breaks into the chromatin using restriction endonucleases, E. coli exonuclease III was used to generate stretches of single stranded DNA. This approach permits detection of the incorporated BrdUrd using anti-BrdUrd monoclonal antibodies. The enzymatic denaturation protocol was optimized using in vitro BrdUrd-labeled L1210 murine leukemia cells, and applied to both in vivo and ex vivo BrdUrd-labeled murine bone marrow cells. With this new method it is possible to study DNA content, cell cycle kinetics and cell surface antigen expression simultaneously, and hence functional relationships between these parameters can be investigated.

Biotinylation of interleukin-2 (IL-2) for flow cytometric analysis of IL-2 receptor expression. Comparison of different methods.

The main prerequisites for the use of biotinylated ligands to study the expression of growth factor receptors on heterogeneous cell populations, such as peripheral blood or bone marrow, by flow cytometric methods, are that the biotinylated ligand retains its binding ability and that binding of the biotinylated ligand to the receptor does not inhibit the subsequent interaction of biotin with fluorescently tagged avidin or streptavidin. Using interleukin-2 (IL-2), we compared the usefulness of various biotinylation reagents, NHS-biotin, S-NHS-biotin, S-NHS-LC-biotin, DBB and photobiotin, and developed optimal biotinylation conditions for the preparation of biologically active biotin-labeled IL-2 and the detection of IL-2 receptor expressing cells by flow cytometry. As determined by spot blot analysis, biotinylation of IL-2 was most efficient at the highest biotin-to-protein (B:P) ratio used. At a B:P ratio of 100, most of the biological activity of IL-2 was retained when S-NHS-LC-biotin was used. In contrast, most of the biological activity of IL-2 samples that were labeled with NHS-biotin or photobiotin was lost under these conditions. Biotin-labeled IL-2 preparations were tested in order to detect IL-2 receptors on IL-2 dependent CTLL-2 cells by flow cytometry after sequential staining with the biotinylated IL-2 and fluorescence tagged streptavidin. A high B:P ratio generally resulted in a high specific fluorescence intensity of the cells, particularly when S-NHS-LC-biotin was used as the biotinylation reagent. Biotin-IL-2 could also be used to detect IL-2 receptors expressed by lymphocytes in peripheral blood and bone marrow. Comparison of staining of lymphocytes with biotinylated IL-2 and an antibody against the IL-2 receptor alpha chain demonstrated that only a subset of the cells that showed a strong fluorescence signal after staining with biotinylated IL-2 expressed high numbers of the IL-2 receptor alpha chain. This is in agreement with the expression of functional IL-2 receptors on resting T cells and NK cells which do not express the alpha chain. After stimulation with PHA, virtually all lymphocytes expressed the alpha chain, whereas only part of these cells showed a strong fluorescence signal after staining with biotin-IL-2, while the rest of the cells had very low numbers of IL-2 binding sites. Our results demonstrate that, in addition to staining individual receptor subunits with antibodies, staining with biotinylated IL-2 is a useful indicator of functional IL-2 receptor expression.

Syntheses of tolrestat analogues containing additional substituents in the ring and their evaluation as aldose reductase inhibitors. Identification of potent, orally active 2-fluoro derivatives.

A series of aldose reductase inhibitors were prepared which were analogues of the potent, orally active inhibitor tolrestat (1). These compounds (5, 7, 9, and 10) have an extra substituent on one of the unoccupied positions on the naphthalene ring of 1. Primary amide prodrugs of several members from the series 5 and 7, namely 6 and 8, respectively, were also prepared. These compounds were evaluated in two in vitro systems: an isolated enzyme preparation from bovine lens to assess their intrinsic inhibitory activity and an isolated sciatic nerve assay to determine their ability to penetrate membranes of nerve tissue. These compounds were also evaluated in vivo as inhibitors of galactitol accumulation in the lens, sciatic nerve, and diaphragm of galactose-fed rats. In general, compounds in series 5, 7, 9, and 10 were potent inhibitors of bovine lens aldose reductase. 2-Halo-substituted analogues from the series 5, 7, and 9 exhibited high activity in the nerve of the 4-day-galactose-fed rat, and in several instances, the primary amide prodrug 8 enhanced the in vivo potency of the respective carboxylic acid 7. Two 2-fluoro-derivatives, 8a and 9a, had especially high activity in vivo and were chosen for additional studies. These compounds were found to be approximately equipotent to tolrestat in the sciatic nerve of the galactose-fed rat and the STZ rat, as judged by their ED50's in these assays. Although primary amide analogue 8a did not have intrinsic inhibitory activity toward aldose reductase, it was metabolized to an active form in vivo and also in vitro within the sciatic nerve.

Discovery of novel non-peptide CCR1 receptor antagonists.

Ligands for the CCR1 receptor (MIP-1alpha and RANTES) have been implicated in a number of chronic inflammatory diseases, most notably multiple sclerosis and rheumatoid arthritis. Because these ligands share a common receptor, CCR1, we sought to discover antagonists for this receptor as an approach to treating these disorders. A novel series of 4-hydroxypiperidines has been discovered by high throughput screening (HTS) which potently inhibits the binding of MIP-1alpha and RANTES to the recombinant human CCR1 chemokine receptor. The structure-activity relationships of various segments of this template are described as the initial HTS lead 1 was optimized synthetically to the highly potent receptor antagonist 6s. This compound has been shown to have at least 200-fold selectivity for inhibition of CCR1 over other human 7-TM receptors, including other chemokine receptors. In addition, data obtained from in vitro functional assays demonstrate the functional antagonism of compound 6s and structurally related analogues against the CCR1 receptor in a concentration dependent manner. The discovery and optimization of potent and selective CCR1 receptor antagonists represented by compound 6s potentially represent a novel approach to the treatment of chronic inflammatory diseases.

Cytochemical hybridization with fluorochrome-labeled RNA. I. Development of a method using nucleic acids bound to agarose beads as a model.

A new procedure to label RNA at the 3'-terminus with a fluorochrome molecule is described. The thiosemicarbazides derived from tetramethylrhodamine isothiocyanate (TRITC) and fluorescein isothiocyanate (FITC) were prepared by reacting these compounds with hydrazine in dimethylsulfoxide (DMSO):pyridine, 99:1 (v/v). They coupled efficiently to the aldehydes generated by periodate oxidation of RNAs. We determined, using Sepharose to which different nucleic acids and proteins had been bound, that the label added no specific binding properties to the RNA, and did not interfere with duplex formation of labeled poly(U) and poly(A). The stability of the fluorochrome-RNA bond under conditions generally used for hybridization was investigated. The bond was found to be unstable at 66 degrees C in 3 x SSC, 0.1% SDS (50% loss within 45 min) but stable for at least 40 hr at 23 degrees C in 70% formamide/3 x SSC. The hybridization characteristics of complementary RNA, both fluorochrome- and 3H-labeled, were investigated using DNA-Sepharose beads as a cytochemical model. Hybridization was measured by scintillation counting of microliter quantities of beads and quantitative fluorescence microscopy of individual Sepharose beads. No influence of the label on the specificity and stability of the hybrids was found. Maximum specific fluorescence was found after hybridization at 23 degrees C in 70% formamide/3 x SSC. These results made possible the successful use of fluorochrome-labeled RNA to perform cytochemical hybridization followed by detection of the hybrids with fluorescence microscopy. This will be described in an accompanying article.

Cytochemical hybridization with fluorochrome-labeled RNA. II. Applications.

The cytochemical detection of specific DNA sequences by hybridization with fluorochrome-labeled RNA and detection of the hybrids by fluorescence microscopy is described. RNAs complementary to the DNA of the kinetoplasts of Crithidia luciliae (an insect trypanosome) or to adenovirus-5 (Ad-5) DNA were labeled with the hydrazine derivative of tetramethylrhodamine isothiocyanate (TRITC). The specificity of the reactions between the complementary RNAs labeled both with 3H and tetramethylrhodamine was studied by cross-hybridization experiments using a model system in which the DNAs were bound to Sepharose beads. The extent of the reaction was measured by scintillation counting of the bead suspensions and quantitative fluorescence microscopy of individual Sepharose beads. The ability of the rhodamine-labeled cRNAs to hybridize and the absence of interference of the fluorochrome label with the specificity of the hybridization reaction was thus demonstrated. After cytochemical hybridization on microscopic preparations of C. luciliae cells the rhodamine-labeled kinetoplast cRNA stains only the kinetoplasts. No fluorescence was observed in the nuclei. After cytochemical hybridization of rhodamine-labeled Ad-5 cRNA with virus infected KB cells a distinct staining pattern in the nuclei was observed. No fluorescence was seen in uninfected cells, or after hybridization with heterologous rhodamine-labeled RNA. The possibilities and limitations of cytochemical hybridization with rhodamine-labeled RNA are discussed.

The development, using poly(Hg-U) in a model system, of a new method to visualize cytochemical hybridization in fluorescence microscopy.

The development, using a model system, of a new method for the detection of cytochemical (in situ) hybrids is described. The method is based on the mercuration of nucleic acids with mercuric acetate. To facilitate hybridization, the acetate ligand is replaced by the CN- ion. In the hybrids formed, the CN- is exchanged for trinitrophenyl(TNP)-glutathione. The TNP-glutathione is subsequently detected by indirect immunofluorescence using anti-TNP antibodies. The feasibility of the approach was investigated using Sepharose- or Sephadex-bound poly(A) and mercurated poly(U). Poly(Hg-U) did hybridize with poly(A)-Sepharose, provided that the acetate ligand was replaced with CN-. The TNP-glutathione hapten thus synthesized bound effectively to mercury-Sepharose but not to amino-Sepharose when the reaction was performed in the dark. Furthermore, binding of TNP-glutathione to Sephadex-bound poly(A) . poly(HG-U) hybrids was detectable with indirect immunofluorescence using anti-TNP antibodies. The fluorescence intensity measured was dependent on the amount of poly(Hg-U) present and on the dilution of the antibody. Nonspecific binding was very low. Calibration of the number of fluorescein molecules found after the complete reaction was performed with fluorescein isothiocyanate-labeled poly(U). It was determined that one fluorochrome molecule per two nucleotides had been obtained, in close agreement with the theoretically expected number. The sensitivity of the method, when applicable to microscopic preparations, is comparable to in situ hybridization with 3H-labeled nucleic acids with a specific activity of 4 x 10(8) dpm/micrograms (two 3H-isotopes per nucleotide) and an exposure time of 1 day. Extension of the method to the cRNA-DNA system and its application to microscopic preparations is under investigation.

Avidin-EITC: an alternative to avidin-FITC in confocal scanning laser microscopy.

Detection of fluorescein-5-isothiocyanate (FITC)-labeled conjugates is suboptimal in two-color confocal scanning laser microscopy (CLSM). This limits the detection of small, dimly fluorescent targets. We explored the possible advantages of applying eosin-5-isothiocyanate (EITC) conjugated to avidin (Av-EITC) as an alternative for Av-FITC in CSLM. Despite the lower quantum efficiency of EITC, we found that the measured Av-EITC and Av-FITC emission intensities were similar as a result of the standard filter combinations used for simultaneous two-color detection in the Bio-Rad MRC 600 CSLM. The advantage of Av-EITC was that its fading characteristics compared very favorably to those of Av-FITC. An excitation intensity-dependent increase in Av-EITC fluorescence was observed, followed by an exponential decrease. This increase in fluorescence allows longer observation times, averaging of several scans without loss of brightness, and thus detection of dimly fluorescent targets by CSLM.

CCR1-specific non-peptide antagonist: efficacy in a rabbit allograft rejection model.

The classic signs of acute cellular rejection during organ transplantation include the infiltration of mononuclear cells into the interstitium. This recruitment of leukocytes into the transplanted tissue is promoted by chemokines like RANTES. Since RANTES is a potent agonist for the CC chemokine receptor CCR1, we examined whether the CCR1 antagonist BX 471 was efficacious in a rabbit kidney transplant rejection model. BX 471 was able to compete with high affinity with the CCR1 ligands MIP-1alpha and RANTES for binding to HEK 293 cells expressing rabbit CCR1. BX 471 was a competitive antagonist of rabbit CCR1 in Ca(2+) flux studies. Two separate studies in which animals were subcutaneously implanted with slow release pellets of BX 471 demonstrated that animals implanted with BX 471 had increased survival compared with untreated controls or animals implanted with placebo. The mean survival time for the placebo group was 12.33+/-1.7 days. The animals in the BX 471 treated group had mean survival times of 16.9+/-2.1 and 16.0+/-1.7 days, respectively, for the two studies. Analysis of the combined data by Student t-test gave a P value of 0.03 that is significant at the 0.05 level. In addition, there was a marked reduction in the urea and creatinine levels in the BX 471 treated animals compared with the control and placebo groups in both studies. Finally, pathologic analysis of the kidneys in the rabbit renal transplantation model from animals in the different groups showed that BX 471 was similar to cyclosporin in its ability to prevent extensive infarction of transplanted kidneys. Based on the data from these studies, BX 471 shows clear efficacy at the single dose tested compared with animals treated with placebo.

Colony-forming cells in chronic granulocytic leukemia--II. Analysis of membrane markers.

Membrane markers and functional properties in vitro of blast cells from the peripheral blood of 2 patients with chronic granulocytic leukemia were studied. Buffy-coat cells were enriched for colony-forming cells by density centrifugation (d less than or equal to 1.062 g cm-3). Upon culture, a large proportion of the (cryopreserved) low-density cells from both patients formed hemopoietic colonies that were heterogeneous with respect to size and cellular composition. Expression of membrane markers on the cells, which had the morphology of undifferentiated blasts, was studied using flow cytometry with a panel of monoclonal antibodies. A striking heterogeneity was observed in that variable numbers of cells were found to express myelomonocytic, megakaryocytic and erythroid membrane markers. Antigenic properties of colony-forming cells were studied by sorting of cells with a fluorescence activated cell sorter. Low numbers of cells (10, 4 and 1, respectively) were sorted directly into the wells of Terasaki microtest plates. With this system, it was shown that myeloid colony-forming cells from patient 1 were exclusively present in HLA-DR-positive cell fractions. Colony formation from the level of a single sorted cell was documented. Sorting of cells labeled with anti-blood-group-H antibody showed that small erythroid colony-forming cells from patient 2 were blood-group-H antigen-positive. These cells did not express HLA-DR. The other colony-forming cells from this patient and essentially all colony-forming cells from patient 1 were HLA-DR-positive and blood-group-H-negative. Although only 2 patients were tested, our studies clearly demonstrate that low-density cell fractions from the blood of patients with CGL provide distinct advantages for the study of membrane properties of hemopoietic cells and of hemopoietic differentiation in general.

Direct morphological and functional examination of murine pluripotent hemopoietic stem cells.

Pluripotent hemopoietic stem cells (PHSC) were isolated from adult mouse bone marrow by a combination of equilibrium density centrifugation and light-activated cell sorting for WGA-positive and H-2k antigen-positive cells. The sorted cells gave rise to 2 spleen colonies per 100 injected cells at 8 days and 6.6 colonies per 100 cells at 12 days after transplantation into lethally irradiated syngeneic recipients. The average enrichment factor for day 12 CFU-S (colony forming unit-spleen) equalled 135 (range, 90-230; n = 15). Enrichment for the cell type that provides radioprotection was equal to 180 +/- 70, indicating that PHSC and CFU-S are identical. Evidence is provided that the spleen seeding efficiency (the f-factor) of these cells was 0.10 and, therefore, that the average purity of the sorted PHSC was 65% (range in 15 experiments, 35-110%). The sorted cells were all in the G0 or G1 phase of the cycle. They appeared to be undifferentiated blasts by morphological criteria, the majority of the cells being similar in structure to the PHSC previously identified in less purified concentrates. 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. Thirty percent of the sorted cells could be induced to form progeny in vitro. This procedure facilitates direct examination of the first events of hemopoietic regulation.

Species selectivity of a small molecule antagonist for the CCR1 chemokine receptor.

The species specificity of a small molecule antagonist for the human CCR1 chemokine receptor, 2-2-diphenyl-5-(4-chlorophenyl)piperidin-1-yl)valeronitrile (CCR1 antagonist 1), has been examined using cloned CCR1 receptors from various species. The compound was able to bind to rabbit, marmoset, and human CCR1, and was able to block the functional activation of these receptors. However, it failed to significantly displace radiolabeled macrophage inflammatory protein-1alpha (MIP-1alpha) binding to mouse CCR1 at concentrations up to 10 microM. These data suggested that the antagonist binding site is well-conserved in rabbit, marmoset and human CCR1, but not in mouse CCR1. The functional selectivity and mechanism of action for CCR1 antagonist 1 were further characterized. CCR1 antagonist 1 blocked the increase in intracellular Ca(2+) stimulated by CCR1 agonists, but had no effect on N-formyl-Met-Leu-Phe (FMLP), monocyte chemotactic protein-1 (MCP-1) and stromal-derived factor 1alpha (SDF1alpha)-induced Ca(2+) mobilization, demonstrating functional selectivity for CCR1. Since CCR1 antagonist 1 is a functional antagonist of marmoset and rabbit CCR1 receptors, it should be possible to test its efficacy in animal models of disease.

Fluorescence microscopical hybridocytochemistry.

A new method has been developed to detect RNA-DNA hybrids in situ by fluorescence microscopy. This overcomes some of the disadvantages of autoradiographical detection of in situ hybridization, notably the low resolution and long exposure times needed. A procedure to label RNA at its 3'-terminus with a fluorochrome molecule has been developed. The optimal conditions for the cytochemical hybridisation reaction of this fluorochrome-labeled RNA were investigated using a model consisting of Sepharose beads to which nucleic acids has been bound. With RNA labeled both with 3H and rhodamine the hybridization reaction could be studied both biochemically by scintillation counting and cytochemically by microfluorometry. The fluorochrome-RNA bond was found to be unstable at higher temperatures. Therefore, the hybridization reaction had to be performed at room temperature in formamide-containing buffers. With fluorochrome-labeled complementary RNA kinetoplast DNA in Crithidia luciliae, adenovirus-5 DNA in infected KB cells and 5S rRNA, tRNA and cloned histone genes in polytene chromosomes of Drosophila could be localised. An immunocytochemical amplification method was developed that increased the sensitivity of the direct method. Several recently developed hybridocytochemical methods based on a combination of a hybridisation reaction in the first layer followed by immunocytochemical second layers, are described. The prospects of the application of the hybridocytochemical techniques in biomedical research, such as gene localisation and virus diagnosis, are discussed.

Flow cytometric detection of beta-globin mRNA in murine haemopoietic tissues using fluorescent in situ hybridization.

The novel method for flow cytometric detection of cellular RNA species in suspended cells by fluorescent in situ hybridization (FC-FISH) was applied in the evaluation of beta-globin expression in murine haemopoietic tissues. Normal murine bone marrow cells and regenerating bone marrow cells obtained after lethal irradiation and bone marrow transplantation as well as murine 15 d fetal liver were examined. Furthermore, spleens and bone marrow of phenylhydrazine-induced anaemic mice were studied. Biotinylated sense- and antisense single strand RNA probes, obtained by transcription of a 510 nucleotides murine beta-globin cDNA sequence subcloned into the pGEM1 plasmid were used as hybridization probes. For detection of the hybrids formed, avidin-FITC was used. Only the antisense beta-globin probe gave strongly positive fluorescence signals in a defined population of cells in each of the tissues examined, whereas the sense probe did not give signals higher than control samples. Melting characteristics of the hybrids showed the specificity of the in situ hybridization reaction. Forward light scatter distributions, reflecting cell size of the positive cells were as expected from erythroid cells. Within the erythrocyte subpopulation both beta-globin-negative and -positive cells were detected. The percentages of positive cells determined flow cytometrically correlated with the percentages observed in May-Grünwald/Giemsa stained preparations. Differences observed in fluorescence intensity between positive cells of different organs were no larger than about a factor of two, indicating a rather constant beta-globin mRNA content over the entire differentiation range. An exception was 15 d fetal liver, which was shown biochemically to contain about eight times more beta-globin RNA and which had a 2.4 times higher fluorescence intensity. We estimate that the sensitivity of the present method is such that as little as 500 copies per cell of a specific mRNA of 1 kb length would be detectable.

Flow cytometric measurement of rRNA levels detected by fluorescent in situ hybridization in differentiating K-562 cells.

The flow cytometric detection of fluorescent in situ hybridization (FISH) performed on intact cells in suspension is a recently described method (Bauman et al. 1989). We studied the application of this method for monitoring cellular differentiation. The amount of rRNA which is taken for a good indicator of growth in size, the rate of protein synthesis and the G0 G1 transition was followed by FISH. For this purpose biotinylated single stranded RNA probes obtained by transcription from a 2.1 kb BglII-EcoRI fragment of the human 28S ribosomal RNA gene subcloned into plasmid pGEM2 were used. K-562 leukaemic cells, used as targets, were induced to differentiate by dimethyl sulfoxide, phorbol myristate acetate and hemin. In the last two cases the cell cycle analysis, growth kinetics, cellular morphology and immunophenotyping indicated differentiation into monocytic and erythroid direction, respectively. The differentiation was accompanied by a rapid increase followed by a decrease to the base level of rRNA. This was not observed in the uninduced exponentially growing control cells. Based on our results, we propose that the FC-FISH detection of the rRNA level is a valuable method to distinguish between cell subpopulations. We propose that using other probes, FC-FISH will become useful to monitor different cellular processes.