Livermore flow cytometer

Peters, David C.; Dean, Phillip N.; Pinkel, Daniel

doi:10.2172/5842852

Cited by 7 publications

(4 citation statements)

References 1 publication

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“…The dual-stained cells were analyzed on the Lawrence Livermore National Laboratory flow cytometer (13). The laser was adjusted to emit approximately 500 mW at 488 nm.…”

Section: Flow Cytometrymentioning

confidence: 99%

Cytochemistry for bromodeoxyuridine/DNA analysis: Stoichiometry and sensitivity

Beisker²,

et al. 1985

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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°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.Key terms: Anti-bromodeoxyuridine, thermal denaturation, stoichiometry, DNA, flow cytometry Several recent reports have described immunochemical procedures in which monoclonal antibodies fluorescently stain for bromodeoxyuridine (BrdUrd) incorporated into cellular DNA (2,3). Ideally, the intensity of fluorescence from immunochemically stained cells is proportional to the amount of incorporated BrdUrd (i.e., the staining is stoichiometric for BrdUrd) and some reports have shown this to be the case in restricted circumstances (2). To the extent that staining is stoichiometric, this new immunochemical procedure may be used as a simple, fast, and accurate alternative to radioactive DNA precursor (i.e., tritiated thymidine) uptake quantification by autoradiog-raphy andor liquid scintillation counting. In addition, Dolbeare et al. (2) have reported a flow cytometric-based immunochemical procedure to stain cells simultaneously for incorporated BrdUrd and relative DNA content. This procedure is particularly useful for studies requiring correlation between cellular DNA content and extent of labeling with a DNA precursor. Such studies include quantification of cell cycle traverse (2,4,5,11,12) and analysis of the kinetic perturbation induced by effects of anticancer drugs (1,2,4,5,12).Most anti-BrdUrd antibodies described so far share the common feature that they bind only to BrdUrd in denatured DNA (3,(14)(15)(16). Thus, in a typical immunochemical procedure, the BrdUrd-labeled cells are fixed, denatured to produce single-stranded DNA, treated with a mouse monoclonal antibody against BrdUrd, washed, treated with a second, fluorescently labeled antimouse antibody, and washed again. Dolbeare et al. (2) showed that by denaturing the DNA partially, the ce...

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“…The dual-stained cells were analyzed on the Lawrence Livermore National Laboratory flow cytometer (13). The laser was adjusted to emit approximately 500 mW at 488 nm.…”

Section: Flow Cytometrymentioning

confidence: 99%

Cytochemistry for bromodeoxyuridine/DNA analysis: Stoichiometry and sensitivity

Beisker²,

et al. 1985

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“…Briefly, cell suspensions in full medium at 37°C were stained with Ho342 from a 500-pM stock solution dissolved in doubly distilled, deionized water. The test tube was rapidly inverted and transferred to the Livermore Flow Cytometer (Peters et al, 1981) modified with a continuous flow 37°C water jacket. The Ho342 was excited with 500-mW UV power (40 mW for sorting experiments) from a Spectra Physics 164-05 argon ion or a 171-01 krypton ion laser (Spectra Physics Inc., Mountain View, CAI, and fluorescence was measured through a 425-nm-long wavelength pass filter.…”

Section: Methodsmentioning

confidence: 99%

FACS analysis of a hyperthermia‐induced alteration in Hoechst 33342 permeability and direct measurement of its relationship to cell survival

Rice

Gray

Dewey

1985

Journal Cellular Physiology

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Heat-induced alterations in CHO-10B cell Hoechst 33342 (Ho342) permeability in vitro were analyzed by flow cytometry. Immediately after 45.5 degrees C heating, uptake was decreased in a dose-dependent manner with cytotoxicity. Kinetic analysis indicated that a treatment that reduced cell survival to approximately 10%, reduced the maximal velocity, Vmax, to 53% of control and increased the dissociation constant, Km, to 156% of control. Also, little change in Ho342 efflux was found to occur from control up to 90 min after heating. Upon incubation at 37 degrees C after the heat treatment from 1 to 24 hr (depending on the severity of the dose) diffuse heterogeneity of Ho342 staining developed which was not evident immediately after heating. The altered staining was not due to the presence of trypan blue staining cells. Membrane permeabilization and nuclei isolation studies indicated that the lesion responsible was most likely a plasma membrane event. Induction of the heterogenous staining was not inhibited by either actinomycin D or hydroxyurea but was inhibited by incubation at 4 degrees C. An inverse correlation existed between Ho342 permeability and clonogenicity, with nearly a 10-fold difference in survival between the high and low fluorescence intensity sorted cells. Also, larger fractions of heat-sensitive S and G2M-phase cells were found in the highly fluorescent sorted fractions. These results are discussed in terms of the putative molecular events that may be involved in hyperthermic modulation of Ho342 permeability.

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“…The dual stained cells were analyzed on the Lawrence Livermore National Laboratory flow cytometer (15). Approximately 5 x lo5 cells per distribution were analyzed.…”

Section: Flow Cytometrymentioning

confidence: 99%

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

et al. 1985

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This report describes a mathematical model of cell proliferation for simulation of bivariate DNAhromodeoxyuridine (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 pM BrdUrd followed by growth in BrdUrdfree medium for 0 to 24 h, or 2) during continuous incubation in 3 pM BrdUrd plus 30 pM 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 (0.081, 7.0 h (0.03, 1.4 h (0.161, and 14.0 h (0.05), respectively. The model is shown to be useful for quantitative interpretation of the bivariate distributions.Key terms: Mathematical model, cell proliferation, cell cycle, DNAhromodeoxyuridine, flow cytometry Quantitative analysis of the cytokinetic properties of normal and neoplastic cell populations is important for determination of the basic growth characteristics of these tissues, for analysis of the cell cycle phase-specific effects of perturbing agents such as anticancer drugs, and to provide a rational basis for cancer therapeutic strategies-especially for the optimal scheduling of chemotherapy and radiation therapy (8). Estimation of cell cycle phase durations, dispersions, growth fraction, etc., for populations under various experimental and therapeutic conditions is usually accomplished by mathematical modeling of experimental data. In this approach, a mathematical model of cell cycle traverse is used to simulate experimental data such as for DNA distribution sequences, fraction of labeled mitosis curves, or bivariate DNA/bromodeoxyuridine (BrdUrd) distribution sequences. The model parameters such as the G1-, S-, and G2M-phase durations, growth fraction, etc., are varied until the simulations match the experimental data as closely as possible. These parameters are then taken as an accurate description of the cytokinetic properties of the cell population under analysis. So far, numerous mathematical models of cell cycle traverse have been proposed (16) and used for simulation of a variety of experimental data (1,7,8,12,17). The validity of these mathematical analyses, of course, depends on the appropriateness of the mathematical model and on the accuracy and information content of the experimental data.The recently developed flow cytometric technique for the simultaneous quantification of total cellular DNA content and incorporated BrdUrd has significantly increased the information available in experimental measurements (3-6). The technique facilitates a rapid, statistically accurate measurement of the distribution (not merely the average) of the amount of BrdUrd incorporated into DNA as a functi...

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Livermore flow cytometer

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Cited by 7 publications

References 1 publication

Cytochemistry for bromodeoxyuridine/DNA analysis: Stoichiometry and sensitivity

Cytochemistry for bromodeoxyuridine/DNA analysis: Stoichiometry and sensitivity

FACS analysis of a hyperthermia‐induced alteration in Hoechst 33342 permeability and direct measurement of its relationship to cell survival

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

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