Laser Flow Microphotometers for Rapid Analysis and Sorting of Individual Mammalian Cells

Mullaney, P. F.; Steinkamp, John A.; Crissman, Harry A.; Cram, L.S.; Holm, D.M.

doi:10.1007/978-1-4615-7323-4_5

Cited by 20 publications

(8 citation statements)

References 51 publications

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“…The corresponding correlation factor is .992. Although the linearity of CVF with the reciprocal light intensity has been already observed by others (12,13), now we can Table 1) versus the reciprocal laser power W. The straight line shown is ohtained with the least-square method (r = ,996). say something more.…”

Section: Resultssupporting

confidence: 61%

Linearity and noise sources in flow cytometry

Ubezio

Andreoni

1985

Cytometry

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A model of pulse formation in flow cytometers is presented that demonstrates the proportionality between the area (or the peak height) of the fluorescence signal produced by the photomultiplier and the number of fluorochrome molecules present in the cell that cause the signal. The model clarifies the possible instrumental origins of inaccuracy in this linearity that results in a broadening of the histograms obtained. A comprehensive formula for the coefficient of variation of the unimodular histograms of an homogeneous population is presented that clearly discriminates among the different contributions of staining, possible inhomogeneity of the examined population, photon statistics, and instrumental instabilities.Finally, some experimental data are presented that show the agreement with the proposed formula.Key terms: Flow cytometry, cellular dye content, fluorescence emission statistics, photomultiplier statistics and noise, propidium iodide stainingIn most applications of flow cytometry, frequency histograms are produced for the content of a given cellular substance (such as DNA, RNA, or proteins) in a population of cells, provided that it is possible to achieve selective and stoichiometric binding of a suitable fluorescent dye to the substance to be examined. The cytometer output is usually a fluorescence frequency histogram as a function of the channel number of registration. To allow this histogram to be interpreted as the distribution of the substance content in the cells, one has to demonstrate that the channel number is linear with the substance content itself. In particular there must be proportionality among the following quantitites: (I) the substance content (X) and the number of bound fluorescent molecules (N); (ID N and the peak height (S) or the area (A) of the fluorescence pulse; (110 the peak height or the area and the channel number (i) of registration.Neglecting the approximations involved in the analogto-digital conversion, point 111 is an obvious consequence of the electronics' linearity; but points I and I1 are not easy to verify (9,10,13,14,16,17). In spite of the fact that much work was done on point I to optimize staining procedures that guarantee the proportionality between N and X (1,4,5,7), point I1 is still under discussion (lo), and its importance in terms of improved measurement precision is underestimated in some experimental work.This paper presents a theoretical discussion of the relation between N and A (or S) and points out the approximations underlying this proportionality. In addition, the lack of proportionality between i and X results in a broadening of the conditional probability P(i/ X), i.e., of the probability of having a count in channel i from a cell with substance content X.Using the results of our linear model in the calculation of the second moment of P(i/X) allows us to show that the instrumental causes of broadening, that is, the inaccuracies of points I1 and I11 above, can become as important as the causes related to the staining procedure of point I. Finally,...

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Section: Resultssupporting

confidence: 61%

Linearity and noise sources in flow cytometry

Ubezio

Andreoni

1985

Cytometry

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“…Complete two parameter analysis of cellular properties also is permitted by using a dual parameter multichannel pulse height analyzer for displaying three-dimensional pulse amplitude distributions . Details concerning instrument and flow chamber design will be discussed elsewhere (17,18) . Fig .…”

Section: Methodsmentioning

confidence: 99%

Rapid, Simultaneous Measurement of Dna, Protein, and Cell Volume in Single Cells From Large Mammalian Cell Populations

Crissman

Steinkamp

1973

The Journal of Cell Biology

514

203

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“…The dissociated spheroid cells were subsequently analyzed as either unfixed cells stained for 5 min with mithramycin in saline just before analysis or as ethanolfixed cells (2-3 hr fiation, followed by a saline wash before staining with mithramycin) using mithramycin concentrations similar to that previously described (4). These mithramycin-stained cells were then analyzed on the Los Alamos Computer-Based Multiparameter Cell Sorter (16) with laser excitation at 457.9 nm. The flow cytometric data acquisition and display programs used have been reported (20).…”

Section: Growthmentioning

confidence: 99%

Correlation of the growth of chinese hamster V₂79‐171b multicellular spheroids with cytokinetic parameters

et al. 1980

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The volumes of Chinese hamster V279-171b multicellular spheroids grown in spinner culture were measured and correlated to DNA distribution and cell volume distribution information. Spheroid growth curves were obtained over a 21-day period using both electronic cell volume measurements and microscope micrometer sizing. The spheroids were collected from the aperture tube after electronic sizing, and single cell suspensions were prepared via trypsinization. Saline-washed cells were then stained with mithramycin and analyzed as unfixed or ethanol-fixed cells for both cell volume and DNA fluorescence measurements. Spheroid growth was determined to have an initial 8-to 9-day rapid growth phase, and a Tumor growth and growth delay measurements as well as cell-cycle measurements have been widely used as tumor biology end points (8,21,23). A better understanding of tumor kinetics might result from detailed correlative information for tumor growth dynamics in relation to cell-cycle kinetic properties of subpopulations comprising the tumor.Multicellular spheroids are of intermediate complexity, between in uztro monolayer cultured cells and subclinical size in uiuo nodular tumors or early metastatic foci. Spheroids are comprised of heterogeneous subpopulations (6,7) and provide a useful cell system for studying growth dynamics by electronic volume (2) ( i e . , Coulter principle) and for monitoring the cytokinetic DNA distribution and cell volume distributions of cells which comprise the spheroids. To date, electronic cell volume, enumeration and sizing (2) of single mammalianPerformed under the auspices of the United States Department of Energy, with joint support from Grant 2258501 from the National Cancer Institute, Department of Health, Education, and Welfare.Presented at Automated Cytology VII, Asilomar, California, November 25-30, 1979. slow phase from 10 to 21 days. The single cell volume distributions of cells obtained from spheroids were dependent on spheroid volume and the relative proportions of small volume noncycling cells to large volume cycling cells comprising the spheroids were also dependent on spheroid volume. DNA distribution data and dual parameter DNA distribution cell volume contour patterns obtained for single cells dissociated from spheroids were closely related to the measured spheroid volume and revealed that the fraction of small, hypoxic Go-like cells was greatest in old, large spheroids.Key terms: Spheroids, cytokinetic, flow cytometry, DNA distributions, cell volume cells have been extensively used and the underlying theory described (1, 2, 12, 24, 25). Conventional Coulter-type apertures having 10-2000 pm diameter orifices are available for single cell and large particle sizing (14). Various reports have described the usefulness of measuring cell volume in relation to cell-cycle information separately (13,15,18,28) and in coincidence with other parameters (3, 12,19,22,27).In the present report, the growth of V279-171b Chinese hamster lung multicellular spheroids were studied and correlated...

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Laser Flow Microphotometers for Rapid Analysis and Sorting of Individual Mammalian Cells

Cited by 20 publications

References 51 publications

Linearity and noise sources in flow cytometry

Linearity and noise sources in flow cytometry

Rapid, Simultaneous Measurement of Dna, Protein, and Cell Volume in Single Cells From Large Mammalian Cell Populations

Correlation of the growth of chinese hamster V₂79‐171b multicellular spheroids with cytokinetic parameters

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Laser Flow Microphotometers for Rapid Analysis and Sorting of Individual Mammalian Cells

Cited by 20 publications

References 51 publications

Linearity and noise sources in flow cytometry

Linearity and noise sources in flow cytometry

Rapid, Simultaneous Measurement of Dna, Protein, and Cell Volume in Single Cells From Large Mammalian Cell Populations

Correlation of the growth of chinese hamster V279‐171b multicellular spheroids with cytokinetic parameters

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Correlation of the growth of chinese hamster V₂79‐171b multicellular spheroids with cytokinetic parameters