Biomedical subjects

A J Beavis

Publications and source records attributed to A J Beavis.

18 recordsLinked to original sources

PubMed2000

Capacitance cytometry: measuring biological cells one by one.

Measuring the DNA content of eukaryotic cells is a fundamental task in biology and medicine. We have observed a linear relationship between the DNA content of eukaryotic cells and the change in capacitance that is evoked by the passage of individual cells across a 1-kHz electric field. This relationship is species-independent; consequently, we have developed a microfluidic technique-"capacitance cytometry"-that can be used to quantify the DNA content of single eukaryotic cells and to analyze the cell-cycle kinetics of populations of cells. Comparisons with standard flow cytometry demonstrate the sensitivity of this new technique.

Animals↗

PubMed1999

Simultaneous analysis of the cyan, yellow and green fluorescent proteins by flow cytometry using single-laser excitation at 458 nm.

BACKGROUND: Development of spectrally distinct green fluorescent protein (GFP) variants has allowed for simultaneous flow cytometric detection of two different colored mutants expressed in a single cell. However, the dual-laser methods employed in such experiments are not widely applicable since they require a specific, expensive laser, and single-laser analysis at 488 nm exhibits considerable spectral overlap. The purpose of this work was to evaluate detection of enhanced cyan fluorescent protein (ECFP) in combination with the enhanced green (EGFP) and enhanced yellow (EYFP) fluorescent proteins by flow cytometry. METHODS: Cells transfected with expression constructs for EGFP, EYFP, or ECFP were analyzed by flow cytometry using excitation wavelengths at 458, 488, or 514 nm. Fluorescence signals were separated with a custom optical filter configuration: 525 nm shortpass and 500 nm longpass dichroics; 480/30 (ECFP), 510/20 (EGFP) and 550/30 (EYFP) bandpasses; 458 nm laser blocking filters. RESULTS: All three fluorescent proteins when expressed individually or in combination in living cells were excited by the 458 nm laser line and their corresponding signals could be electronically compensated in real time. CONCLUSIONS: This method demonstrates the detection of three fluorescent proteins expressed simultaneously in living cells using single laser excitation and is applicable for use on flow cytometers equipped with a tunable argon ion laser.

Cells, Cultured↗

PubMed1999

An integral membrane green fluorescent protein marker, Us9-GFP, is quantitatively retained in cells during propidium iodide-based cell cycle analysis by flow cytometry.

Previously, we described GFP-spectrin, a membrane-localized derivative of the green fluorescent protein that can be employed as a marker during the simultaneous identification of transfected cells and cell cycle analysis by flow cytometry (Kalejta et al., Cytometry 29: 286-291, 1997). A membrane-anchored GFP fusion protein is necessary because the ethanol permeabilization step required to achieve efficient propidium iodide staining allows cytoplasmic GFP to leach out of the cell. However, viable cells expressing GFP-spectrin are not as bright as cells expressing cytoplasmic GFP and their fluorescence intensity is further diminished after ethanol treatment. Here, we demonstrate that the fluorescence intensity of cells expressing an integral membrane GFP fusion protein (Us9-GFP) is similar to that of cells expressing cytoplasmic GFP and is quantitatively maintained in cells after ethanol treatment. By allowing an accurate assessment of the expression level of GFP, Us9-GFP allows a more precise analysis of the effects of a cotransfected plasmid on the cell cycle and thus represents an improvement upon the original membrane-associated GFP fusion proteins employed in this assay.

Cell Cycle↗

PubMed1999

Differential expression of integrin subunits in canine knee ligament fibroblasts.

A method for measuring the expression of integrin subunits on the cell surface of knee ligament fibroblasts was developed with use of flow cytometry and immunofluorescence. The ligament cells exhibited uniform size and density, as shown by forward and side-scatter properties, and showed minimal nonspecific binding of isotype control antibodies compared with unstained cells. All cells expressed the alpha5 integrin subunit; lateral collateral ligament cells stained with antibody to alpha5 showed a mean fluorescence intensity 2-fold higher than that of medial collateral ligament cells, 1.5-fold higher than that of posterior cruciate ligament cells, and 3-fold higher than that of anterior cruciate ligament cells, indicating a greater expression of the alpha5 subunit by lateral collateral ligament cells than by medial collateral, posterior cruciate, and anterior cruciate ligament cells. All cells expressed the beta1 integrin subunit; the expression by posterior cruciate ligament cells was 3-fold higher than that by medial collateral ligament or lateral collateral ligament cells and 5-fold higher than that by anterior cruciate ligament cells. All cells expressed the beta3 integrin subunit; the expression by posterior cruciate ligament cells was 1.5, 3, and 4.5-fold greater than that by lateral collateral, anterior cruciate, and medial collateral ligament cells, respectively. Our data suggest there is a differential expression of integrin subunits in knee ligament fibroblasts, and this in part may explain differences in their attachment and adherence to extracellular matrix molecules.

Animals↗

PubMed1997

Use of a membrane-localized green fluorescent protein allows simultaneous identification of transfected cells and cell cycle analysis by flow cytometry.

The simultaneous detection of the green fluorescent protein (GFP) and DNA content using propidium iodide (PI) by flow cytometry is made difficult because of the unique nature of these 2 fluorogenic reagents. For PI to enter cells efficiently and to stain DNA quantitatively, the cells must first be permeabilized; ethanol treatment is a routine method to achieve this. However, this permeabilization step causes GFP, which is normally found in the cytoplasm, to leach out of the cells. Although the use of paraformaldehyde-based fixatives allows GFP to be maintained in cells and retain its fluorescence even after ethanol permeabilization, the protocol we commonly employ results in inefficient PI staining and poor quality DNA histograms. To circumvent these difficulties, we have employed a GFP-fusion protein which localizes to the cellular membrane and as such is retained in cells upon ethanol permeabilization without prior fixation. This allows the GFP signal to be detected in cells treated with ethanol in preparation for PI staining and cell cycle analysis. This property facilitates the use of GFP as a marker for transfected cells in experiments designed to characterize the effects of ectopic expression of cellular or viral genes on cell cycle progression.

Biomarkers↗

PubMed1997

Flow cytometric quantitation of attachment and phagocytosis in phenotypically-defined subpopulations of cells using PKH26-labeled Fc gamma R-specific probes.

Human receptors for IgG (Fc gamma R) are characterized by diverse structure and function. We describe a flow cytometric technique to quantitate receptor-specific Fc gamma R-mediated attachment and phagocytosis in phenotypically-defined subsets of cells using erythrocytes (E) labeled with the lipophilic dye PKH26 and coupled with biotin/avidin to either human IgG (myeloma proteins) or anti-Fc gamma R mAb. Using this technique and Fc gamma RIIa as a model, (1) we demonstrate sensitive and specific quantitation of attached and internalized E coupled to receptor-specific mAb or natural ligand by monocytes within a peripheral blood leukocyte preparation; (2) we show the capacity to detect subtle allelic differences in Fc gamma R function; and (3) we demonstrate oxidant-induced enhancement of binding and internalization.

Cell Adhesion↗

PubMed1996

Allo-7: a new fluorescent tandem dye for use in flow cytometry.

This report describes the development of a novel tandem dye by combining allophycocyanine (APC) and cyanine dye indotricarbocyanine (CY7) to create ALLO-7 for use in flow cytometry. The APC donor fluorophore was excited at 647 nm and, through resonance energy transfer to the CY7 acceptor, produced fluorescence at > 780 nm. To test the applicability of this tandem in single and multicolor immunofluorescence, a streptavidin conjugate of the tandem (SA-ALLO-7) was used for the detection of cell surface antigens on human peripheral blood leukocytes (PBL) by indirect immunofluorescence. Human PBL were stained with CD4/ GaM-APC, CD3-fluorescein isothiocyanate (FITC), CD14-phycoerythrin (PE), CD19-energy-coupled dye (phycoerythrin-Texas Red) (ECD), and CD8-biotin with SA-ALLO-7 and analyzed for fluorescence on a FACS Vantage using dual-laser excitation (488 and 647 nm). The results indicated that the percentage of cells positive for each of the surface antigens was comparable for single-color controls and multicolor samples. The ALLO-7 fluorescence, which was collected with a 730-shortpass dichroic mirror and a 790/50-bandpass filter, was clearly resolved from the APC fluorescence and that from FITC, PE, and ECD. The SA-ALLO-7 exhibited minimal nonspecific binding to PBL monocytes. However, the specific binding of the tandem to high-density antigens was clearly identified by positive fluorescence. This unique tandem reagent, ALLO-7, provided the capability for dual-color immunofluorescence with a 647-nm laser line (or a helium neon laser at 633 nm) and provides the potential to perform three-color analysis with a dye-head laser (Texas Red, APC, ALLO-7).

Antibodies, Monoclonal↗

PubMed1996

Detection of cell-surface antigens using antibody-conjugated fluorospheres (ACF): application for six-color immunofluorescence.

Antibody-conjugated fluorospheres (ACF) were used to phenotype murine leukocytes by flow cytometric analysis. Multicolor immunofluoresence (beyond simultaneous 4-color analysis) is limited by the availability of specific antibody-fluorochrome chrome conjugates and even further restricted by the spectral emission overlap of many of the fluorochromes when used in combination. Fluorospheres possessing unique excitation/emission spectra can provide much needed versatility to existing protocols of multicolor fluorescence. SKY BLUE (647 nm excitation, 730 nm emission) fluorospheres conjugated to CD11b monoclonal antibody were used in combination with the monoclonal antibodies IAd-FITC, L3T4 (CD4)-PE, LYT2 (CD8)-APC, THY1.2 (CD90)-biotin and B220 (CD45R)-RED613 for the simultaneous detection of six distinct cell-surface antigens in a mixed cell population. All fluorescence signals were resolved, and comparison of results from five-, six- and single-color samples indicated that the percentages of cells positive for specific surface antigens were equivalent.

Animals↗

PubMed1995

T cells are necessary for Th2 cytokine production and eosinophil accumulation in airways of antigen-challenged allergic mice.

In a murine model of pulmonary inflammation, aerosolized antigen challenge of sensitized B6D2F1 mice leads to eosinophil accumulation within the lungs. Little is known of the role of T cells and their cytokine products in these allergic animals. In this study, we show that T cells migrate into the lungs in response to antigen challenge and are necessary for local production of cytokines (IL-4 and IL-5) important in B and T cell development as well as eosinophil activation and differentiation. Flow cytometry revealed an increase in the percentage of Thy1+ T cells but not in B220+ B cells in bronchoalveolar lavage fluid after challenge when compared to unchallenged mice. Although there was an increase in both T cell subsets, there were twice as many CD4+ cells as CD8+ cells at 24 hr and after 48 hr the CD4+ subset predominated. The CD4+ T lymphocytes were CD44+ CD45RBlo indicating an activated/memory phenotype and tracheobroncheal lymph node cells obtained from challenged mice proliferated in a dose-dependent manner in response to antigen stimulation in vitro. Reverse transcriptase-polymerase chain reaction analysis of lung tissue-derived RNA indicated an increase in Th2-like cytokines. IL-4 and IL-5 steady-state mRNAs were at peak levels 6 hr after challenge, while no consistent increase was found for IFN-gamma mRNA levels. Treatment with the glucocorticoid betamethasone just prior to challenge reduced the levels of cytokine mRNA as well as the eosinophil influx. In vivo depletion of T cells from sensitized mice reduced pulmonary eosinophilia as well as the expression of IL-4, IL-5, and IFN-gamma steady-state mRNAs in the lungs of sensitized and challenged mice. These results indicate that T cells migrating into the lungs of mice after antigen challenge play an important role in the production of Th2-like cytokines and the accumulation of eosinophils in bronchial fluids.

Animals↗

PubMed1994

Simultaneous measurement of five-cell surface antigens by five-colour immunofluorescence.

Multicolour immunofluorescence and flow cytometry were used for simultaneous measurement of five-cell surface antigens on murine spleen cells. We have been able to quantitate T-cells, T-cell subsets, B cells, and expression of the activation marker I-Ad from a single sample using four directly conjugated monoclonal antibodies LYT2-APC, L3T4-PE, B220-RED613, I-Ad-FITC and one indirect step THY1.2-biotin/streptavidin-Cascade Blue. Three excitation wavelengths were used (488 nm, 647 nm, and U.V. 351-364 nm) for fluorescence measurements. The combination of fluorochromes used provided good resolution such that all five fluorescence signals were spectrally resolved. The percentage of cells positive for expression of a specific cell surface marker were almost identical for single-colour samples and the five-colour analysis, differing by only 0.3-1.5 percentage points.

Animals↗

PubMed1994

Tracking of murine spleen cells in vivo: detection of PKH26-labeled cells in the pancreas of non-obese diabetic (NOD) mice.

Flow cytometry was used to track the in vivo migration of PKH26-labeled donor spleen cells from diabetic NOD mice that were injected into non-diabetic recipient NOD mice. Flow cytometric analysis of recipient mouse tissues revealed that the donor cells were present in the peripheral blood, spleen and lymph nodes 24 h following injection and could still be detected after 28 days. PKH26(+) cells were also detectable in the pancreas 7 days after injection. Phenotypic analysis of the PKH26(+) cells that migrated into these target organs and tissues showed that the major cell population detected was Thy1.2(+) T-lymphocytes, predominantly the Thy1.2(+)/L3T4(+) subpopulation, but Thy1.2(+)/Lyt2(+) cells as well as B220(+) cells (B lymphocytes) were also present.

Animals↗

PubMed1994

Suppression of the allogeneic response by human IL-10: a critical role for suppression of a synergy between IL-2 and TNF-alpha.

The effect of IL-10 on T-cell activation by alloantigens in primary mixed lymphocyte reaction (MLR) was examined. IL-10 strongly suppressed proliferation and cytokine synthesis observed in this reaction. To determine the cytokine synthesis inhibition that was critical for the IL-10 induced suppression of proliferation in MLR, the effect of exogenous cytokines (IL-1 alpha, IL-1 beta, IL-2, IL-4, IL-6, IFN-gamma, or TNF-alpha) on this suppression was examined. None of these cytokines, when used at high concentration, was able to completely restore proliferation in the MLR to the levels observed in the absence of IL-10. However, IL-2 and TNF-alpha, when added alone at high concentration, could partially overcome the IL-10 induced suppression of proliferation in MLR. Moreover, when a combination of IL-2 and TNF-alpha was added at suboptimal doses to IL-10-suppressed MLR, complete restoration of the proliferative response was obtained. The ability of IL-10 to suppress proliferation in MLR was dependent on the type of cells used as stimulators. Thus, IL-10 suppressed proliferation in MLR when allogeneic normal peripheral blood mononuclear cells (PBMC), highly purified monocytes or B cells, were used, but not when B-cell lines were used as stimulators. Investigation of the effect of IL-10 on cytokine synthesis revealed that when B-cell lines were used as stimulators of MLR, IL-10 suppressed IFN-gamma and IL-2 synthesis but was unable to suppress TNF-alpha production. In contrast, CSA, which inhibited proliferation in MLR induced by B-cell lines, also inhibited TNF-alpha. IL-2 and IFN-gamma synthesis. Together these data suggest that the suppression of MLR by IL-10 requires the effective inhibition of both IL-2 and TNF-alpha production to suppress a synergy between these two cytokines.

Antibodies, Monoclonal↗

PubMed1993

Differential induction of stromelysin mRNA by bovine articular chondrocytes treated with interferon-gamma and interleukin-1 alpha.

Articular chondrocytes from rheumatoid joints have been shown to express class II major histocompatibility (MHC) antigens that were correlated with the presence of interferon-gamma (IFN-gamma) in the inflamed joint. Chondrocytes expressing MHC antigens function as antigen presenting cells and thus stimulate lymphocyte proliferation. These responses suggest a powerful role for the IFN-gamma stimulation of chondrocytes. The present studies were designed to examine the functional role of chondrocytes exposed to IFN-gamma during cartilage degradation that occurs in synovial disease. Destruction of cartilage in arthritis is partially attributable to metalloproteinases released by the chondrocytes in response to interleukin-1 (IL-1). Bovine articular chondrocytes treated with interleukin-1 alpha (IL-1 alpha) produced enhanced levels of stromelysin mRNA, however, Northern blots could not determine the percentage of cells responding. Exposure of bovine articular chondrocytes to IFN-gamma induced the expression of bovine HLA-DR (boHLA-DR) antigen in 50% of the cells. Using a modified cell sorting technique, chondrocytes that expressed class II MHC antigens produced two fold greater stromelysin mRNA than chondrocytes that did not express this antigen. In contrast, collagen type II mRNA levels were similar in chondrocytes, regardless of the expression of class II MHC antigens. In situ hybridization studies showed that less than half of all cartilage chondrocytes were induced to synthesize stromelysin mRNA. These observations suggest that IFN-gamma stimulates specific subpopulations of chondrocytes to be functionally active in inflammation-induced metalloprotease secretion.

Animals↗

PubMed1991

Enhanced hydroperoxide production by peripheral blood leukocytes following exposure of murine epidermis to 12-O-tetradecanoylphorbol-13-acetate.

Previous studies have shown that the phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulates phagocytic leukocytes to produce reactive oxygen intermediates in vitro. The present studies focused on the production of reactive oxygen intermediates by peripheral blood leukocyte cell populations following in vivo exposure of murine epidermis to TPA. TPA induced a dose-dependent (0.2-20 micrograms) increase in polymorphonuclear cells (PMN) that appeared to be recruited from the marginal pools, while simultaneously decreasing the number of peripheral blood mononuclear cells. These alterations were detected as early as 2 h following topical application of TPA and persisted over a 21 day time period, using a twice-weekly TPA treatment schedule. The oxidation of 2',7'-dichlorofluorescin (DCFH) was used to examine the hydroperoxide production in peripheral blood PMN isolated from SENCAR mice treated with TPA. TPA stimulated a 2-fold increase in PMN-associated DCFH oxidation (645.4 +/- 118 fmol DCF) 4 h after topical application of 10 micrograms TPA when compared to PMN isolated from acetone-treated mice (339.0 +/- 35.8 fmol DCF). These observations suggest that topical application of TPA recruits PMN that are activated prior to their infiltration into the epidermis. Given the ability of these cells to migrate to local sites, they may serve as a primed cell population that significantly contributes to cutaneous alterations observed during acute and chronic inflammation following TPA exposure.

Administration, Cutaneous↗

PubMed1990

Production of hydrogen peroxide by murine epidermal keratinocytes following treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate.

The ability of murine epidermal cells to produce intracellular hydrogen peroxide was analyzed by flow cytometry and the measurement of 2',7'- dichlorofluorescin (DCFH) oxidation. Epidermal cells isolated from acetone-treated CD-1 mice for 24 h were relatively homogeneous in cell size and density and oxidized low levels of DCFH. However, following 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment of mice (10 micrograms; 24 h), two cytokeratin-positive populations of cells were identified that were heterogeneous with respect to size and density. These two TPA-derived cell populations oxidized levels of DCFH that were time and dose dependent and were between 2- and 10-fold higher than levels of DCFH oxidized by cells isolated from acetone-treated mice. The ability of catalase, the enzyme that detoxifies hydrogen peroxide, to suppress DCFH oxidation to control levels suggested that intracellular hydrogen peroxide was responsible for the enhanced rate of DCFH oxidation in epidermal cells isolated from TPA-treated mice. The ability of mouse epidermal keratinocytes to oxidize DCFH in response to TPA treatment was confirmed using a cloned keratinocyte cell line. These results suggest that specific subpopulations of keratinocytes produce elevated levels of intracellular peroxides following treatment with TPA either in vivo or in culture.

Animals↗

PubMed1990

Identification of epidermal cell subpopulations with increased ornithine decarboxylase activity following treatment of murine epidermis with 12-O-tetradecanoylphorbol-13-acetate.

Ornithine decarboxylase (ODC), the initial enzyme in the polyamine biosynthetic pathway, has been used as a marker for the hyperplasia that occurs following exposure of mouse epidermis to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Using flow cytometry in combination with polyclonal antibodies to ODC, we examined the levels of ODC-associated immunoreactive protein present within mouse epidermal cells at 4 and 24 h after a single topical application of TPA, as well as following chronic exposure to TPA and in papillomas. Basal levels of ODC-specific antibody binding were detectable in acetone-treated CD-1 mouse epidermis and were increased 3-fold at 4 h after TPA treatment. The amount of ODC antibody binding detected after exposure to 17 nmol TPA twice weekly for 3 weeks was similar to that detected within cells isolated from papillomas and was 2.5-fold higher than in cells isolated at 4 h after a single topical treatment of mice with TPA. These observations support the hypothesis that specific subpopulations of keratinocytes constitutively express high levels of ODC following chronic exposure to TPA. The novel method for ODC detection described in these studies provides a means to identify, isolate, and further characterize epidermal cells that may give rise to papillomas and carcinomas.

Animals↗

PubMed1990

Differentiation of U-937 histiocytic lymphoma cells towards mature neutrophilic granulocytes by dibutyryl cyclic adenosine-3',5'-monophosphate.

Treatment of U-937 cells with the cyclic nucleotide analog, dibutyryl cyclic adenosine-3',5'-monophosphate (dBcAMP) induced these cells to differentiate towards granulocytes. dBcAMP produced a dose- and time-dependent inhibition of U-937 cell growth reaching a maximum after 48-h treatment with 500 microM. At this concentration, dBcAMP had no effect on cell viability. Treatment with dBcAMP caused a rapid (within 24 h) decrease in the number of cells in the S phase of the cell cycle, with a concomitant increase in cells in the G0/G1 phase. dBcAMP also induced the appearance of f-met-leu-phe receptors on U-937 cells as well as the ability to produce hydrogen peroxide and superoxide anion. These data suggest that dBcAMP-treated U-937 cells were functionally mature. Using specific monoclonal antibodies and flow cytometry, we found that differentiated U-937 cells expressed the monocytic/granulocytic surface markers MY8 and MAC-1, but not the monocyte specific markers MO2 or MY4. In addition, dBcAMP-treated U-937 cells did not stain for nonspecific esterase, displayed less HLA-DR antibody binding than undifferentiated cells and appeared smaller and more granular. These are all characteristics of mature granulocytes. Taken together our studies indicate that differentiation of U-937 cells is not necessarily limited to the monocytic pathway of development.

Antibodies, Monoclonal↗

PubMed1990

Flow cytometric detection of ornithine decarboxylase activity in epidermal cell subpopulations.

Using flow cytometry in combination with membrane permeabilization techniques to enhance binding of antibodies with immunoreactive protein within the cytoplasm, we have developed a method to examine the ornithine decarboxylase (ODC) activity present within subpopulations of epidermal cells following acute and chronic exposure to the phorbol ester tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA). The method described has the sensitivity to detect basal levels of ODC as well as increases in ODC at early time points following treatment with TPA and has the additional advantage of allowing subpopulation identification and characterization.

Animals↗