Cell Microfluorometry: A Method for Rapid Fluorescence Measurement

Dilla, M. A. Van; Truiullo, T. T.; Mullaney, P. F.; Coultex, J. R.

doi:10.1126/science.163.3872.1213

Cited by 439 publications

(120 citation statements)

References 9 publications

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“…These cells are advantageous because growth of wild-type populations is inhibited by cAMP, and cAMP-insensitive mutants can be derived that are defective in the cAMP binding protein and its associated protein kinase (17,18). In the present studies we have used the flow-microfluorimeter (19) phosphate-buffered saline and fixed in phosphate-buffered saline containing 10% Formalin. After DNA was stained with acriflavin (21), the cells were analyzed in the Lawrence Livermore Laboratory flow-microfluorimeter; the apparatus is described in detail elsewhere (22).…”

mentioning

confidence: 99%

Cyclic AMP, a nonessential regulator of the cell cycle.

Coffino

Gray

Tomkins

1975

Proc. Natl. Acad. Sci. U.S.A.

101

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Flow-microfluorimetric analysis has been carried out on populations of exponentially growing S49 mouse lymphoma cells treated with dibutyryl cyclic AMP./The drug produces a specific concentration-dependent kblock in the GI phase of the cell cycle while other phases of the cycle are not perceptibly altered. The cell cycle of a line of mutant cells lacking the cyclic AMP-dependent protein kinase is not affected by the drug. Since these mutant cells have been shown to maintain a normal cell cycle, even in the presence of high levels of cyclic AMP, periodic fluctuations in the levels of the cyclic nucleotide cannot be required for or determine progression through the cell cycle.There is considerable evidence that adenosine 3': 5'-cyclic monophosphate (cAMP) has a regulatory effect on the growth of cells in culture. The levels of the cyclic nucleotide increase as untransformed cells approach confluency (1,2), although this is contradicted by others (3). Intracellular cAMP concentrations are negatively correlated with growth rate among a variety of fibroblast cell lines (4). Transformed cells have a lower cAMP content than untransformed cells (1, 5). Exogenous cAMP analogs or the induction of endogenous cAMP slows or stops growth of some cells (6-10). Proliferation of contact-inhibited cells, induced by refeeding or proteolytic treatment, is prevented by cAMP analogs (7,11). The cAMP level changes during the cell cycle and is specifically low in mitosis (11,12).While these results make it clear that cAMP can cause marked effects on the cell cycle, important questions remain unanswered.(i) Is cAMP merely a negative regulator of the cycle, or is variation of cellular cAMP levels a necessary signal that entrains the cell cycle? Burger et al. (11) have proposed, for instance, that a fall in cAMP is the signal that necessarily precedes DNA synthesis.(ii) What is the locus of the growth-inhibitory action of cAMP? This has been variously reported as lying in G1 (7,13,14), in G2 (8), or in multiple discrete portions of the cycle (15, 16). These studies have in most cases used cell populations synchronized by techniques that possibly cause unbalanced growth, which might raise questions regarding the use of these materials for examining the effect of a growth regulatory substance.To answer these questions we have studied growth regulation in cultured S49 mouse lymphoma cells. These cells are advantageous because growth of wild-type populations is inhibited by cAMP, and cAMP-insensitive mutants can be derived that are defective in the cAMP binding protein and its associated protein kinase (17,18). In the present studies we have used the flow-microfluorimeter (19)

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mentioning

confidence: 99%

Cyclic AMP, a nonessential regulator of the cell cycle.

Coffino

Gray

Tomkins

1975

Proc. Natl. Acad. Sci. U.S.A.

101

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“…In one mouse liver experiment, a 6C cell peak appeared in the flow cytometry histogram, but a direct measurement of DNA content by absorption cytometry failed to detect cells with such a peak. We therefore believe that some caution may be warranted in the use of flow cytometry alone for evaluation of DNA distributions and of the proportions of different types of cells in complex solid tissues.Key terms: Absorption cytometry, flow cytometry, DNA content, cell ploidy It was also found that the coefficients of variation (CVs) in the DNA measurements of GO/G1 tumor cells made by flow cytometry (12,20) were, in general, lower than those of the results reported when similar cells were measured with the early absorption cytometry techniques (22). The rapidity and ease of performance of flow cytometry, coupled with the relatively low GO/ G1 CVs (which were attributed to increased "accuracy"), soon established flow cytometry as the standard method for quantitating cellular DNA content.…”

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confidence: 99%

“…Key terms: Absorption cytometry, flow cytometry, DNA content, cell ploidy It was also found that the coefficients of variation (CVs) in the DNA measurements of GO/G1 tumor cells made by flow cytometry (12,20) were, in general, lower than those of the results reported when similar cells were measured with the early absorption cytometry techniques (22). The rapidity and ease of performance of flow cytometry, coupled with the relatively low GO/ G1 CVs (which were attributed to increased "accuracy"), soon established flow cytometry as the standard method for quantitating cellular DNA content.…”

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Differences in the flow and absorption cytometric DNA distributions of mouse hepatocytes and tumor cells

et al. 1989

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We have determined the DNA content, the ploidy levels, and the percentages of different cell types present in small and large mouse mammary tumors as well as in young and old mouse livers by using absorption and flow cytometry. Absorption cytometry data indicated a significant increase in the proportion of transformed GO/G1 cells in the tumors as compared to that of the stromal GO/Gl cells with progressive tumor growth. This increase was not detected by flow cytometry. In both young and old mouse livers, a small number of cells of higher ploidy (8C and 1 6 0 were detected by absorption cytometry but were not apparent in histograms obtained by flow cytomRecently, there has been considerable interest in the quantitative analysis of cellular DNA content for investigations as to whether the ploidy levels and cell cycle distributions of malignant tumors correlate with clinical outcome (6)(7)(8)13,14,18). Early studies performed with absorption cytometry showed that aneuploid DNA content was associated with malignancy (6,10,11). Such measurements, however, were time consuming and often inaccurate because of optical errors in the measuring system (10,111. With the introduction of flow cytometry, measurements of the DNA content in cells of a large number of samples from different sources have become much more rapid. In this technique, cells or nuclei are stained with fluorochromes such as propidium iodide (PI) or ethidium bromide (EB), which fluoresce in direct proportion to the amount of double-stranded nucleic acid present. The samples are then analyzed for the amount of fluorescence per particle. The relative amount of DNA per cell is usually calculated by reference to the fluorescence of a known internal standard (17,21).etry. Furthermore, changes in the proportions of liver cells of different ploidy with age were apparent in absorption cytometry data but not in flow cytometry data. In one mouse liver experiment, a 6C cell peak appeared in the flow cytometry histogram, but a direct measurement of DNA content by absorption cytometry failed to detect cells with such a peak. We therefore believe that some caution may be warranted in the use of flow cytometry alone for evaluation of DNA distributions and of the proportions of different types of cells in complex solid tissues.Key terms: Absorption cytometry, flow cytometry, DNA content, cell ploidy It was also found that the coefficients of variation (CVs) in the DNA measurements of GO/G1 tumor cells made by flow cytometry (12,20) were, in general, lower than those of the results reported when similar cells were measured with the early absorption cytometry techniques (22). The rapidity and ease of performance of flow cytometry, coupled with the relatively low GO/ G1 CVs (which were attributed to increased "accuracy"), soon established flow cytometry as the standard method for quantitating cellular DNA content. There was still concern by some, however, that errors, caused by measurement of nonviable debris, could lead to false peaks or other gross errors in the DNA distri...

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“…Two projector lenses were originally used as relay optics (1:l magnification) to focus the image of the laser intersection point onto a PMT (34). The first lens collected the emitted fluorescence and focused it at infinity through a filter to the second lens, which refocused the light onto a PMT pinhole aperture.…”

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confidence: 99%

A modular detector for flow cytometric multicolor fluorescence measurements

1987

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A modular detector for measuring multicolor fluorescence from cells illuminated by single or multiple lasers has been developed for flow cytometers. Motion picture projector, camera, and CCTVlvideo lenses were evaluated for use in the detector by comparing their physical characteristics, image quality, and light collection efficiencies. A 25-mm focal length F/0.95 CCTV lens was selected, based on our critera and test results. The detector was constructed out of square aluminum extrusion channels. A CCTV lens mounted on the outside of the first channel collected light emitted from cells and collimated it towards filters and secondary CCTV lenses located in each channel. The secondary lenses functioned as relay optics for directing and focusSeveral optical systems have been designed for measuring light scatter and fluorescence in flow cytometers (FCM). Early methods employed bright-fieladark-field microscopic on-axis measurements for quantifying absorption, light scatter (12), and fluorescence (11). In these detection systems, high numerical aperture (NA) objective lenses were used to magnify and image the cell stream onto apertures in front of photomultiplier tubes (PMTs). Dichroic color-separating filters were used to divide the absorption, light scatter, and fluorescence. A similar dark-field illumination instrument for quantifying fluorescence and light scatter was constructed around a 10 x low-power microscope (27).Epiilluminators (15) have been used to measure fluorescence (5,241 and light scatter (23) and they have been used with flow chambers that have electronic particlevolume sensing (9) and cell-sorting (13) capability. High-NA objectives serve as both the excitation and emission collection lens. The illuminating light is reflected by a n excitation dichroic mirror and imaged onto the cell stream. The longer-wavelength fluorescence emission is then collected by the same lens and transmitted by the dichroic mirror for imaging onto PMT detectors. Multibeam, sequential excitation also has been used to illuminate cells for measuring absorption, scatter, and ing light onto pinhole spatial filters for measurement by photomultipliers. The detector design allowed any number of channels to be connected together and the versatility for making simultaneous or sequential measurements. Measurements on lymphocytes labeled with four monoclonal antibodies conjugated to fluorescent dyes and measurements on viable tumor cells stained for DNA content and with three fluorescent-labeled antibodies were used to demonstrate the detector's capabilities.Key terms: Fluorescence detector, immunofluorescence, lymphocytes, tumor-cell analysis multicolor fluorescence (3). Recently, the fluorescence emission spectra of cells have been recorded using a monochromator connected to a vertical-illuminator flow cytometer (25).The orthogonal dark-field method uses a lens placed perpendicular to the laser-beadcell-stream axes to collect the emitted fluorescence and light scatter. Two projector lenses were originally used as relay optics (1:l...

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Cell Microfluorometry: A Method for Rapid Fluorescence Measurement

Cited by 439 publications

References 9 publications

Cyclic AMP, a nonessential regulator of the cell cycle.

Cyclic AMP, a nonessential regulator of the cell cycle.

Differences in the flow and absorption cytometric DNA distributions of mouse hepatocytes and tumor cells

A modular detector for flow cytometric multicolor fluorescence measurements

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