Flow cytometric analysis of recombinant murine GM-CSF (rmuGM-CSF) induced changes in the distribution of specific cell populations in vivo

The changes in the distribution of granulocytes, monocytes, and lymphocytes in various tissue compartments following subcutaneous (SC) administration of recombinant murine GM-CSF (rmuGM-CSF) in vivo was determined by flow cytometry in time course studies. Balb/c mice were given single, daily SC injections of 1 or 4 micrograms of rmuGM-CSF for 10 days. Flow cytometric analysis was performed on bone marrow (BMC), peritoneal exudate (PEC), and peripheral blood (PBC) cell preparations from mice treated for 1, 3, and 10 days. Dual fluorescence was employed to gate on leukocytes (T200+) and analyze for Ig+, Thy 1.2+, MAC+, and 8C5+ (granulocytes) cells. The analyses indicated that SC-rmuGM-CSF increased the percentage of 8C5+ cells in PBC after 1 day of treatment. However, significant changes in the cell composition of PEC and BMC were not observed until day 10 of treatment and included increases in 8C5+ cells and the myeloid cell population, respectively. Side scatter analysis (cell density) of PBC and PEC indicated that the percentage of the granulocytic cell population increased significantly in rmuGM-CSF treated mice. The changes observed in PEC and BMC appeared to be dose-related whereas those observed in PBC were not. These data clearly demonstrate the utility of flow cytometric analyses for detecting selective effects of cytokines on cell populations that are involved in host defense mechanisms.     

Association of 5′nucleotidase activity with immature granulocytes in the bone marrow of guinea pigs

Bone marrow leukocytes from adult strain 2 guinea pigs were found to have appreciable levels of 5′adenosine monophosphate hydrolytic activity (105 nmole/h/10⁶ cells). On the basis of substrate specificity studies, enzyme inhibition studies, and thin-layer chromatographic analysis of the reaction product, the activity is related to 5′nucleotidase (5′N). The enzyme activity was associated with the membrane-enriched particulate fraction of lysed bone marrow cells.The bone marrow cell-associated 5′N activity was consistently very high in all five strains of guinea pigs examined (77–127 nmole/h/10⁶ cells) and the range of activity was at least 10-fold greater than that observed for bone marrow cells obtained from mice, rabbits or rats. Furthermore, the bone marrow cell-associated 5′N activity in strain 2 guinea pigs was 5-fold greater than that observed for spleen and at least 13-fold greater than that of blood, mesenteric lymph nodes or thymus obtained from the same animal.Fractionation of strain 2 guinea pig bone marrow cells on Percoll density gradients showed that as the proportion of immature granulocytes increased in the various cell fractions, so did the 5′N activity. The cell fraction that sedimented at a density of 1.071 g/ml had the highest 5′N activity and the majority of the cells (94%) were immature granulocytes. The bone marrow compared to blood and spleen had the highest number of total granulocytes and the highest percentage of immature granulocytes. We conclude that the elevated bone marrow-derived 5′N activity in guinea pigs is associated with the resident population of immature granulocytes in that tissue.     

HISTOCHEMICAL DEMONSTRATION OF DEHYDROGENASE ACTIVITY IN THE CELLS OF NORMAL HUMAN BLOOD AND BONE MARROW

Endogenous and succinic dehydrogenase activity was demonstrated in the living cells of normal human blood and bone marrow using a buffered nitro BT-succinate incubating solution. With this technique dehydrogenase activity was localized primarily in the granular leukocytes and the sites of enzymatic activity appeared to be non-mitochondrial. The addition of a non-ionic surface active agent to the incubating solution resulted in marked differences in the cellular and intracellular localization of dehydrogenase activity. With this method it was possible to demonstrate dehydrogenase activity in the mitochondria of most of the formed elements of the blood and bone marrow, including developing granulocytes and erythroid cells, agranulocytes, and blood platelets. Mature erythrocytes also exhibited a minimal dehydrogenase reaction with this procedure. This investigation indicated that in order adequately to demonstrate and evaluate dehydrogenase activity in the cells of the blood and bone marrow it was necessary to have increased cellular and mitochondrial permeability, as well as partially viable cells with an intact dehydrogenase system.     

A novel multiparameter flow cytometric assay for inosine triphosphatase expression analysis in leukocytes

The aim of this study was to assess inosine triphosphate (ITPase) expression in the different leukocyte populations present in peripheral blood samples of a nonimmune compromised control group. For this purpose, a multiparameter flow cytometric assay was developed and performed to study ITPase expression in peripheral leukocyte subpopulations of healthy volunteers (n = 20). Qualitative ITPase expression was assessed by determining the percentage of ITPase-positive cells. Quantitative data were obtained by measuring the median fluorescent intensity (MFI). Subcellular localization of ITPase was analyzed using immunocytochemistry. Immunocytochemistry showed that ITPase is present in all leukocytes and localized intracellular. Based on this finding, a multiparameter flow cytometric assay was developed using a Fix & Perm strategy. Qualitative and quantitative ITPase expression remained stable (variation, <10%) for at least 48 h after blood sampling. MFI values showed that activated monocytes contained significantly more ITPase when compared to the total monocyte fraction (P < 0.0001), which subsequently had a higher amount of expression than granulocytes (P < 0.0001). In addition, the phagocyte subpopulations ([activated] monocytes and granulocytes) contained significantly higher levels of ITPase when compared to lymphocytes (P < 0.0001). Within the lymphocyte fraction, it appeared that T-helper cells contained significantly higher ITPase levels when compared to cytotoxic T cells, B lymphocytes, and natural killer cells (P < 0.0001). Our study is the first which describes a flow cytometry assay to analyze ITPase expression in leukocytes qualitatively as well as quantitatively and visualizes the intracellular localization of ITPase in leukocytes. ? 2012 International Society for Advancement of Cytometry.     

Characteristics of the dynamics of hematopoietic precursor cells cloned in diffusion chambers and recorded in experiments on mice and dogs irradiated in median lethal doses]

In experiments with mice and dogs irradiated with LD50, it was shown the postirradiation depopulation of haemopoietic polypotent (CFUs) cell-precursors in mouse bone marrow was more pronounced than that of granulocytic and macrophagal cells (CFUdc). The rate of repopulation of CFUs during the first week was higher than that of CFUdc (T1/2 was 2.5 and 8.8 days respectively). In dogs, one could notice a partial change in the colony formation, a prolonged plateau period in the postirradiation CFUdc dynamics, and a coincidence in time with cellularity restoration in the bone marrow and peripheral blood leukocytes. It is suggested that in conditions of heterogeneous incubation in diffuse chambers, the haemopoietic cell-precursors are more mature than in the syngeneic system. The method of CFUdc determination has proved to be ineffective in estimating the onset and intensity of the postirradiation haemopoiesis recovery in dogs. The study of the bone marrow CFUdc population may, however, be used in intact animals to predict the probability of their death after irradiation within the median lethal dose range.     

The repopulating potential and differentiation capacity of hematopoietic stem cells from the blood and bone marrow of normal mice

Using the hematopoietic colony technique, we have investigated the repopulating potential of bone marrow cells and leukocytes of blood from normal mice and have demonstrated that the frequency of hematopoietic stem cells in bone marrow is 50 to 150 times that of stem cells in the circulating blood. The differentiation capacity of these stem cells has also been examined. Results of comparative studies of the serial sections of hematopoietic colonies formed from marrow and blood leukocytes indicate that the differentiation capacity of stem cells from marrow and blood is similar, and that at least 80% of these cells differentiate along a single cell line. Thus, peripheral blood stem cells can effect a complete hematopoietic graft, establishing in the host, donor red cells, granulocytes, and platelets. The possibility that blood leukocytes may serve as a potential source of stem cells for hematopoietic transplants has been considered. Although blood contains stem cells, their frequency is so low as to make it unlikely that they would become a useful source of precursor cells for transplantation purposes.     

Augmentation of activities of peripheral granulocytes and of resistance against bacterial infection after administration of G-CSF into mice]

We evaluated the metabolic capability of murine peripheral granulocytes after administration of recombinant human granulocyte colony-stimulating factor (rhG-CSF) by quantitative flow cytometric assay for H2O2-dependent oxidative product formation. Intraperitoneal administration of a daily dose of 10 micrograms of rhG-CSF for 5 days induced doubling of the leukocyte population. Differential counting of peripheral leukocytes and scattergram by flow cytometry showed an increased mature granulocyte population. After stimulation with phorbol myristate acetate, the granulocytes of the rhG-CSF-administered mice demonstrated some hyperresponsive population and an increased H2O2 production. The hyperresponsive population showed H2O2 production 4-6 times higher than did normal cells. Granulocytes from the G-CSF-treated mice revealed an augmented phagocytic activity and an increased expression of Mac-1 molecules. Moreover, mice treated with G-CSF showed an enhanced resistance against intravenous infection with a lethal dose of E. coli. Granulocytes showing such markedly increased oxidative metabolism may be a significant component of the host defence to various infective organisms.     

Flow-cytometric characterization of stimulation, free radical formation, peroxidase activity and phagocytosis of human granulocytes with 2,7-dichlorofluorescein (DCF)

A standardized four-step assay for the flow cytometric determination of the oxidative activity of human polymorphonuclear leukocytes (PMNL) from normal human individuals and from septic patients was developed, using 2,7-dichlorofluorescin-diacetate (DCFH-DA) as indicator for the intracellular formation of H2O2 and free radicals. Spontaneous H2O2 and free radical formation was measured by preincubation of buffy coat PMNLs from fresh peripheral venous blood at 37 degrees C and pH 7.4 with 10 microM DCFH-DA. Intracellular peroxidase activity was determined by addition of 1 mM external H2O2 to this assay. A maximum of granulocyte oxidative burst activity was elicited by the addition of 150 nM phorbol-myristate-acetate (PMA). A physiological burst was generated by incubating buffy coat PMNLs together with E. coli bacteria. The DNA of dead cells was in all instances simultaneously counterstained with propidium iodide (PI). Quiescent or H2O2 or bacteria treated granulocytes moved as a single cell cluster to higher fluorescences. Stimulation with PMA, in contrast, generated always a bimodal distribution of granulocyte fluorescence with the high activity cell cluster being approximately sevenfold more active than the low activity cell cluster. Roughly half of the granulocytes in normal individuals had high fluorescence. An increase of the high activity granulocytes was observed in septic patients. Model experiments with the nonfluorescent DCFH-DA cleavage product DCFH (2,7-dichlorofluorescin) showed that DCFH was quickly photo-oxidized to fluorescent DCF (2,7-dichlorofluorescein) by UV-light and to a lower degree by daylight. DCFH even slowly autooxidized in the dark.(ABSTRACT TRUNCATED AT 250 WORDS)     

绿色荧光转基因大鼠模型的建立

目的随着干细胞研究的推进,大鼠干细胞的研究日趋迫切。本研究旨在为活体荧光影像系统、干细胞归巢、细胞移植体内示踪研究,提供绿色荧光蛋白EGFP转基因大鼠模型。方法通过显微注射方式获得EGFP转基因大鼠,采用活体荧光影像系统、激光共聚焦显微镜,对EGFP转基因大鼠各个组织的荧光表达水平进行比较;采用流式细胞术检测转基因大鼠血液和骨髓细胞、骨髓干细胞的荧光标记率,筛选骨髓干细胞高效标记绿色荧光的转基因大鼠。结果建立了心脏、肝脏、肌肉、肺、胰腺、脑、膀胱、胃、肾脏、肠和脾脏组织中,系统性表达EGFP的SD-TgN（ACT-EGFP-1）ZLFILAS转基因大鼠;流式细胞术检测表明,该品系血液细胞绿色荧光标记率为94.4%,骨髓干细胞绿色荧光标记率为97.8%。结论建立了多组织系统性高表达绿色荧光,骨髓干细胞荧光标记率高达95%以上的转基因大鼠,为影像分析,造血干细胞的归巢等研究提供了大鼠模型。     

Asialogangliosides as differentiation markers of rat erythropoietic and granulopoietic cells in rat bone marrow. Flow cytometric analysis of rat bone marrow cells using anti-asialoganglioside antibodies

Flow cytometric analysis using anti-glycolipid antiserum was used on rat bone marrow cells to determine the relation between the glycolipid species expressed on cell surfaces and cell differentiation. Four kinds of antibodies against gangliotriaosylceramide (Gg3Cer), gangliotetraosylceramide (Gg4Cer), fucogangliotetraosylceramide (IV2 alpha Fuc-Gg4Cer) and IV3 alpha Gal-fucogangliotetraosylceramide (IV3 alpha GalIV2 alpha Fuc-Gg4Cer, blood group B lipid) were used. The cells sorted out by each anti-glycolipid antiserum were stained with May-Grünwald-Giemsa reagent and identified by microscopy. In the erythropoietic group, only polychromatic erythroblasts had these four glycolipids on their cell surfaces; none appeared on differentiated erythrocytes. These glycolipids were expressed during the early stages of immature granulocytes, especially in the promyelocyte and myelocyte stages of eosinophilic and neutrophilic granulocytes. Very limited populations of lymphocytes were sorted out as asialoganglioside-expressing cells. We concluded that asialogangliosides are useful differentiation markers for the erythropoietic and granulopoietic cells of rat bone marrow, and that anti-asialoganglioside antibody-flow cytometry is a very useful technique with which to isolate immature granulocytes and erythropoietic cells from rat bone marrow cells.     

Flow cytometric assay for the measurement of human bone marrow phenotype, function and cell cycle

A flow cytometric assay for the measurement of human bone marrow and blood leukocyte antigen expression, phagocytosis, and proliferation is described. Subpopulations of leukocytes were identified by their light scatter characteristics, and the expression of a myeloid differentiation antigen (designated CDw65) determined following incubation with CDw65 specific fluorescein-isothiocyanate (FITC) conjugated monoclonal antibodies (VIM2). Incubation of leukocytes with ethidium monoazide (EMA) labeled Candida albicans followed by staining with FITC conjugated VIM2 allowed the combined determination of cellular CDw65 expression and phagocytic capacity. In addition, immunostained leukocytes were fixed, and their DNA labeled with propidium iodide (PI), before CDw65 expression was measured for cells in different phases of the cell cycle. The method allows evaluation of phenotypic and functional heterogeneity, as well as cell cycle parameters, within subpopulations of cells during hematopoietic differentiation.     

Direct flow cytometric quantification of alkaline phosphatase activity in rat bone marrow stromal cells.

Cell preparations in cytochemistry are conventionally analyzed with transmitted light after fixation and reaction with agents such as azo-coupling dyes. With cell suspensions stained with fluorescent cytochemical dyes, cells can also be analyzed and sorted by flow cytometry. We have exploited the intense red fluorescence of Fast Red Violet LB generated in cytochemical reactions to perform flow cytometric analyses of alkaline phosphatase (AP) expression in rat bone marrow stromal cells. By modifying staining protocols of single-cell suspensions, we demonstrate that in comparison to staining with Fast Red TR, the method is specific, can distinguish among various levels of enzyme expression within the whole population, and permits enzyme kinetic studies of heterogeneous cell populations. The method was applied to study the effect of the glucocorticoid dexamethasone (Dx) on cell proliferation and AP expression. In low AP-expressing cells, Dx treatment at 10(-8) M increased the [3H]-thymidine labeling index from 3.85% to 5.24% (p less than 0.01). In contrast, high AP-expressing cells were unlabeled by [3H]-thymidine. The staining and analytical methods reported here facilitate the detection, isolation, and quantification of subpopulations of bone marrow stromal cells that express alkaline phosphatase activity. These experiments demonstrate the value of flow cytometry as an adjunct to conventional cytochemical methods.     

Flow cytometric analysis of micronucleated reticulocytes in mouse bone marrow

This laboratory has previously reported a flow cytometric procedure for quantitatively analyzing mouse peripheral blood reticulocytes for micronucleus content. The current study extends this line of investigation by evaluating whether these same flow cytometric scoring procedures can be applied to the analysis of mouse bone marrow samples. To validate the method, three groups of male BALB/c mice were treated with 100 mg/kg b.wt. methyl methanesulfonate. Bone marrow samples were collected 20, 40 or 60 h after administration. A set of 5 untreated animals was included to provide an indication of spontaneous micronucleus frequencies. The cells were fixed with ultracold methanol, treated with ribonuclease, and labeled with anti-CD71 antibody (FITC conjugate) and propidium iodide. This fixing and labeling procedure resulted in the resolution of the micronucleated reticulocyte population and facilitated high-speed acquisition and enumeration via flow cytometry. The number of micronucleated reticulocytes was determined flow cytometrically by the analysis of 10?000 total reticulocytes per bone marrow sample. In addition to these automated measurements, slides stained with acridine orange were prepared and the number of micronuclei per 1000 reticulocytes was determined microscopically for each sample. The resulting data demonstrate that flow cytometry can effectively enumerate micronucleated reticulocytes in mouse bone marrow. The advantages associated with an objective, high throughput scoring methodology are also clearly indicated.     

Stimulation of differentiation and proliferation of haemopoietic cells in vitro

A liquid culture system, for haemopoietic cells, has been developed using bone marrow cells alone, or co-cultures of thymus and bone marrow cells, inoculated into four ounce medical bottles. After several days growth, such cultures consisted of an attaching population of cells, forming discrete colonies, and a non-attaching population. In the (co-cultures) there was a 2 X enhancement of monolayer colony development compared with the combined total present in the (marrow alone) plus (thymus alone) cultures. Also, better maintenance of non-attaching cells was seen in the (co-cultures). Normal CFUS and CFUC were present in both the (marrow alone) and the (co-cultures) for at least 14 days. In the (marrow alone) cultures, granulocytes in all stages of development were present for the first week, but by 12 days the culture consisted mainly of mono-nuclear cells. In the (co-cultures), however, at 12 days more than 60% of the cells were granulocytes, in all stages of differentiation. (Co-cultures) established using lethally irradiated thymus cells were not able to support this prolonged myeloid differentiation. By feeding the (co-cultures) it was possible to maintain production of (granulocytic) cells for at least ten weeks, although no fully mature granulocytes were observed. After the second feeding, no CFU_S were detectable, but variable numbers of agar colony forming cells (not classical CFU_C) were present at least for ten weeks.     

Collection of hematopoietic stem cells from canine peripheral blood and their ability to regenerate hematopoiesis]

A procedure is presented for the collection of a large number of hemopoietic stem cells from the peripheral blood of dogs by means of a single leukapheresis using the NCI-IBM Blood Cell Separator. In the course of a leukapheresis of about 285 min duration a mean of 23 x 10-9 leukocytes is collected from the blood. The hemopoietic stem cells among such separated leukocytes initiate repopulation of bone marrow within 10 days after whole body X-irradiation with 1200 R. The cell numbers in a defined histological section of femoral bone marrow are evaluated 9 to 10 days after irradiation and subsequent autologous transfusion of 6.72 x 10-9 separated mononuclear leukocytes. The results indicate that the bone marrow cell numbers of transfused dogs are significantly greater than in dogs given only 1200 R and reach a level of approximately 49% of the normal value. Possible ways of increasing the yield of hemopoietic stem cells from the peripheral blood will be considered.     

Quaternary benzo[c]phenanthridine alkaloids--novel cell permeant and red fluorescing DNA probes.

BACKGROUND: Quaternary benzo[c]phenanthridine alkaloids (QBAs) are naturally occurring compounds isolated from plants in the Fumariaceae, Papaveraceae, Ranunculaceae, and Rutaceae families. In addition to having a wide range of biological activities, they are also attractive for their fluorescent properties. We observed interesting fluorescent characteristics in the QBAs-macarpine (MA), sanguirubine (SR), chelirubine (CHR), sanguilutine (SL), chelilutine (CHL), sanguinarine (SA) and chelerythrine (CHE) after interaction with living cells. METHODS: Water stock solutions of the alkaloids (10-100 microg/ml) were added to intact cells, and after a brief incubation the cells were observed. Human cell lines HL60 (human promyelocytic leukemia), HeLa (human cervix adenocarcinoma), and LEP (human lung fibroblasts), and piglet blood were used in the experiments. Blood cells were stained with MA in combination with FITC-conjugated anti-CD45 surface marker antibody. Cells were analyzed by fluorescence microscopy and by flow cytometry. RESULTS: All tested alkaloids immediately entered living cells with MA, CHR, and SA binding to DNA. MA showed the best DNA staining properties. Fluorescence microscopy of MA, CHR, and SA stained cells described the nuclear architecture and clearly described chromosomes and apoptotic fragments in living cells. Moreover MA can rapidly represent the cellular DNA content of living cells at a resolution adequate for cell cycle analysis. QBAs were excitable using common argon lasers (488 nm) emitting at a range of 575-755 nm (i.e. fluorescence detectors FL2-5). Spectral characteristics of MA allow simultaneous surface immunophenotyping. CONCLUSIONS: It was shown that MA, CHR, and SA stain nucleic acids in living cells. They can be used as supravital fluorescent DNA probes, both in fluorescence microscopy and flow cytometry, including multiparameter analysis of peripheral blood and bone marrow. MA binds DNA stochiometrically and can provide information on DNA content.     

Simultaneous flow cytometric analysis of surface markers and nuclear Ki-67 antigen in leukemia and lymphoma

The monoclonal antibody Ki-67 identifies an antigen present during the late G1, S, G2, and M phases of the cell cycle, whereas resting cells do not express this antigen. Immunostaining with Ki-67 provides a simple method with which to determine the growth fraction of a malignant cell population without requiring a laborious procedure or use of radioactive materials. Thus far, detection of Ki-67-positive cells by flow cytometry was limited because of nuclear location of the antigen. In this study, periodate-lysine-paraformaldehyde (PLP) fixation of cells in suspension, labeling with Ki-67, and the subsequent flow cytometric analysis of the tumor growth fraction is described. Fixation with PLP at -10 degrees C for 15 min rendered the plasma membrane permeable without destroying cell surface antigens. Thus double immunofluorescence studies using both a surface marker and Ki-67 could be performed. This offers the additional advantage of being able to define the phenotype of proliferating cells. This method was applied to determine the growth fraction in peripheral blood and bone marrow samples of patients with leukemia and non-Hodgkin's lymphoma. The results of Ki-67 studies in 91 patients are shown. A wide variability of individual Ki-67 values was observed within each entity. Use of this flow cytometric procedure substantially facilitates the quantification of proliferating cells in pathological blood and bone marrow samples.