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Biomedical subjects

F Dolbeare

Publications and source records attributed to F Dolbeare.

15 recordsLinked to original sources
PubMed1989

Flow cytofluorometric analysis of enzyme reactions based on quenching of fluorescence by the final reaction product: detection of glucose-6-phosphate dehydrogenase deficiency in human erythrocytes.

We developed a method for accurate cytofluorometric analysis of the final reaction product of enzyme reactions in individual cells. Glucose-6-phosphate dehydrogenase (G6PD) activity in human erythrocytes was demonstrated cytochemically, and the amount of final reaction product (formazan) per cell was detected indirectly by quenching of autofluorescence generated by glutaraldehyde fixation. Formazan quenches fluorescence in a dose-dependent manner. The method has been used for detection of G6PD deficiency. Heterozygous and homo(hemi)zygous deficiency could easily be established, even in cases of extreme "Lyonization" where microscopic inspection failed to discriminate between either normal individuals and heterozygously deficient patients or heterozygously and homozygously deficient patients. The principle of quenching of fluorescence by final reaction products of enzymes can be applied to flow cytofluorometric analysis of enzyme activity in individual cells in general.

Dose-Response Relationship, Drug↗
PubMed1988

Use of restriction endonucleases and exonuclease III to expose halogenated pyrimidines for immunochemical staining.

We describe an enzymatic procedure for exposure of single-stranded DNA (ssDNA) containing the halogenated pyrimidines (HdUrd) bromodeoxyuridine (BrdUrd) or iododeoxyuridine (IdUrd) in single cells to antibodies that bind to HrdUrd only in ssDNA. Production of ssDNA was accomplished by digesting the DNA using either restriction endonucleases alone or endonucleases followed by exonuclease III. The enzymatic production of ssDNA was maximal when 0.1 N HCl or 0.1 M citric acid plus Triton X-100 was added to extract nuclear proteins prior to enzymatic denaturation. The restriction endonucleases Bam HI, Dde I, Eco RI, and Hind III produced significant ssDNA when used alone to allow binding of detectable amounts of the anti-HdUrd antibody IU-4 in Chinese hamster ovary cells labeled with 10 microM BrdUrd or 10 microM IdUrd. However, these treatments did not expose sufficient ssDNA to allow binding of IU-1, an anti-HdUrd antibody with lower binding affinity. IU-4 binding was most intense after treatment with Eco RI. Treatment with exonuclease III following endonuclease digestion allowed substantially more IU-4 binding.

Animals↗
PubMed1987

An improved immunocytochemical procedure for high-sensitivity detection of incorporated bromodeoxyuridine.

This report describes an improved immunochemical procedure to stain cells in suspension for incorporated bromodeoxyuridine (BrdUrd) and total DNA content. The procedure consists of five steps: chromatin proteins are extracted by treating with 0.1 M HCl and 0.7% Triton X-100 to facilitate DNA denaturation and to minimize nonspecific staining; cellular DNA is denatured by heating to 100 degrees C in distilled water; BrdUrd in single-stranded DNA (ssDNA) is stained using an immunochemical procedure; autofluorescence is reduced using sodium borohydride (NaBH4); and DNA is stained with the fluorescent dye propidium iodide. With this procedure, the BrdUrd incorporated by CHO cells during periods as short as a few seconds can be detected using flow cytometry. In addition, the stoichiometry of the immunofluorescent staining procedure is high.

Animals↗
PubMed1986

Improved high-affinity monoclonal antibody to iododeoxyuridine.

Three monoclonal antibodies (Mabs), IU-1, IU-4, and B-44 were evaluated in enzyme immunoassays (ELISA) and by flow cytometry for their abilities to recognize bromodeoxyuridine (BrdUrd)- and iododeoxyuridine (IdUrd)-substituted DNA's, nucleotides, and nucleosides. IU-4 is a new Mab, derived from mice immunized with 5-iodo-2'-deoxyuridine-5'-monophosphate (IdUMP) conjugated (IdUMP) conjugated through the phosphate group to albumin. This immunogen was selected to resemble IdUMP in DNA. In competition ELISA assays, IU-4 prefers IdUrd to BrdUrd and prefers halogenated nucleotides over the corresponding nucleosides. In both ELISA and flow analysis, IU-4 recognizes IdUrd in DNA at substitution frequencies at least as low as one IdUrd one per 1,000 normal bases. The high affinity of IU-4 for IdUrd-DNA contrasts with IU-1 and B-44, which show a strong binding dependency on the frequency of base substitution and require DNA that is essentially fully substituted with BrdUrd for binding in both flow and ELISA assays. The high affinity of IU-4 for IdUrd in DNA and its independence of IdUrd residue spacing make it a superior reagent for the quantitative labeling of halogenated thymidine analogues in whole cells.

Animals↗
PubMed1986

Expression of proliferating cell nuclear antigen (PCNA)/cyclin during the cell cycle.

The expression of proliferating cell nuclear antigen (PCNA), also called cyclin, was quantified in the cell lines SP2/0 and MOLT-4 and in mouse splenocytes induced to proliferate in vitro with mitogens. Autoantibody from a patient with systemic lupus erythematosus was used to label PCNA in cell suspensions after the cells had been fixed and permeabilized. In the same cells DNA was stained by propidium iodide. The cells were then analysed by flow cytometry for PCNA and DNA content. The PCNA profiles in proliferating spleen cells and the cell lines were similar. Most G0-G1 cells did not express significant amount of PCNA. A dramatic increase in PCNA immunofluorescence was observed in late G1 cells, and further increases were observed in S-phase cells. G2-M cells showed a reduced level of PCNA immunofluorescence relative to S-phase cells but were still elevated relative to G0-G1 cells. Proliferating cells arrested at the G1-S boundary by exposure to cytosine arabinoside showed an increased PCNA immunofluorescence as compared to unstimulated cells.

Animals↗
PubMed1986

Cell cycle analysis using flow cytometry.

This manuscript reviews the utility of flow cytometry for the study of cell proliferation. The applications of univariate DNA distribution analysis to cytokinetic studies of asynchronous and perturbed cell populations are discussed briefly. The newly developed technique for simultaneous flow cytometric measurement of cellular DNA content and amount of incorporated bromodeoxyuridine is discussed in more detail. The cytochemistry required for this analysis is reviewed as are its applications to: determination of the fractions of cells in the G1-, S- and G2 + M-phases of the cell cycle; determination of the G1-, S- and G2 + M phase durations and dispersions and growth fraction for asynchronous cells; detection of ara-C resistant cells present at low frequency in an otherwise sensitive population; and analysis of the cytokinetic response of a solid murine tumour to treatment in vivo with a cell cycle specific agent.

Animals↗
PubMed1985

Cytochemistry for bromodeoxyuridine/DNA analysis: stoichiometry and sensitivity.

This report describes an improved immunochemical procedure for staining cells in suspension for amount of incorporated bromodeoxyuridine (BrdUrd) and total DNA. In this procedure, cellular DNA is partially denatured by extracting the cells with 0.1 M HCl and then heating them to 80 degrees C in a 50% formamide solution. The cells are then immunofluorescently stained using a monoclonal antibody against BrdUrd in single-strand DNA (ssDNA) and counterstained for DNA content with propidium iodide (PI), a dye that fluoresces preferentially when bound to double-strand DNA (dsDNA). We show that the relative amounts of immunofluorescently stained BrdUrd in ssDNA and PI in dsDNA can be altered reciprocally by changing the formamide concentration, denaturation time, and denaturation temperature. We show that this new immunochemical staining procedure allows more complete DNA denaturation so that fivefold lower levels of BrdUrd incorporation can be quantified. In addition, we show that the BrdUrd-linked immunofluorescence achieved using the new denaturation procedure is more linearly related to cellular BrdUrd content than that achieved after acid DNA denaturation. However, cell loss is sufficiently severe with the thermal denaturation procedure that it may not be applicable to all cell types.

Animals↗
PubMed1985

Cell cycle analysis using numerical simulation of bivariate DNA/bromodeoxyuridine distributions.

This report describes a mathematical model of cell proliferation for simulation of bivariate DNA/bromodeoxyuridine (BrdUrd) distributions. The model formulates the change with time in the frequency of cells with any DNA content and in the amount of incorporated BrdUrd, according to given cytokinetic parameters, i.e., durations and dispersions of cell cycle phases and DNA synthesis rate during S-phase. We have applied this model to sequential DNA/BrdUrd distributions measured for Chinese hamster ovary cells asynchronously grown in vitro, 1) for 30 min in 10 microM BrdUrd followed by growth in BrdUrd-free medium for 0 to 24 h, or 2) during continuous incubation in 3 microM BrdUrd plus 30 microM thymidine for 2 to 24 h. The matches between the experimental and simulated distributions give the G1, S, G2M, and total cell cycle durations (and coefficients of variation) of 5.6 h (0.08), 7.0 h (0.07), 1.4 h (0.16), and 14.0 h (0.05), respectively. The model is shown to be useful for quantitative interpretation of the bivariate distributions.

Animals↗
PubMed1985

Detection of cytosine arabinoside resistant cells at low frequency using the bromodeoxyuridine/DNA assay.

This report describes a rapid and sensitive procedure for detection of cytosine arabinoside- (Ara-C) resistant mouse leukemia cells (L1210) in a predominantly Ara-C-sensitive population. L1210 cell lines sensitive or resistant to Ara-C were grown and treated with Ara-C in vitro or in vivo. Ara-C-resistant cells were detected as those cells with S-phase DNA content retaining the ability to incorporate bromodeoxyuridine (BrdUrd) after treatment with Ara-C. The BrdUrd incorporation ability of the S-phase cells was assessed by simultaneous flow cytometric measurement of cellular DNA content and amount of incorporated BrdUrd. The proportion of Ara-C-resistant cells was accurately estimated at frequencies approaching 10(-3).

Animals↗
PubMed1979

Flow microfluorometric identification of liver cells with elevated gamma-glutamyltranspeptidase activity after carcinogen exposure.

We have developed a fluorometric cytochemical assay for gamma-glutamyltranspeptidase (gamma-GT) using the substrate gamma-glutamyl-4-methoxy-2-naphthylamide in which the released methoxynaphthylamine was coupled with 5-nitrosalicylaldehyde to form a yellow fluorescent crystalline product within the cells. Single cell suspensions were obtained by collagenase perfusion of livers from rats that had either received a two-thirds partial hepatectomy followed 24 hr later by a single injection of diethylnitrosamine (DEN) or received a partial hepatectomy alone. Cultured HTC cells were used as a source of gamma-GT+ cells. Fluorescence (gamma-GT activity) was low in most of the cells from both DEN-exposed and control rats, but high in HTC cells. The livers of both DEN-exposed and control rats had a subpopulation of cells that were gamma-GT+; this population could be quantitated and sorted by flow cytometry. Five weeks post injection the number of GT+ cells from the rats exposed to DEN was more than 20 times that from the control rats. Increased gamma-GT activity may be a useful cytochemical marker for preneoplastic liver cells.

Animals↗
PubMed1979

A fluorescent assay of proteinases in cultured mammalian cells.

We have demonstrated proteinase activity in unfixed cells grown on tissue culture plates with a technique using 5-nitrosalicylaldehyde and peptide derivatives of 4-methoxy-2-naphthylamine. The 4-methoxy-2-naphthylamine liberated by proteinase activity reacts with 5-nitrosalicylaldehyde to form a fluorescent product. The substrates CBZ-alanyl-arginyl-arginyl-4-methoxy-2-naphthylamine and lysyl-alanyl-4-methoxy-2-naphthylamine, were used for the direct visual detection of two arylamidase activities in BALB/c 3T3 and C3H 10T 1/2 cells. With low magnification these enzyme activities can be detected in single clones; with higher magnification the fluorescent product can be seen within the cytoplasm of single cells.

Animals↗
PubMed1979

Dynamic assay of enzyme activities in single cells by flow cytometry.

Three enzymes in single cells were assayed dynamically by flow cytometry using four fluorogenic substrates. Acid phosphatase was determined with 7-bromo-3-hydroxy-2-naphtho-o-anisidine (naphthol AS-BI) phosphate and 4-methylumbelliferone (MU) phosphate, neutral esterase with fluorescein diacetate, and lactic dehydrogenase with NAD-sodium lactate. Fluorescence measurements obtained with the flow cytometer were converted into relative specific enzyme activities for single cells with molar fluorescence coefficients determined with a spectrofluorometer. Specific activities obtained from spectrofluorometric data were compared with activities calculated from flow cytometeric data. Flow cytometric assays gave lower specific single cell activities for 4-methylumbelliferone phosphate hydrolysis and for lactic dehydrogenase than did similar assays by standard spectrofluorometry. Product diffusion may be the greatest cause for this discrepancy.

Acid Phosphatase↗