High resolution chromosome analysis: one and two parameter flow cytometry

Gray, J. W.; Langlois, Richard G.; Carrano, A.V.; Burkhart-Schulte, K.; Dilla, M. A. Van

doi:10.1007/bf00294840

Cited by 125 publications

(38 citation statements)

References 23 publications

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“…Figure 7 is a bivariate distribution showing the performance of HiSS in resolving human chromosomes 9-22 at a measurement rate of about 15,000 s-'. This performance is comparable to that obtained in conventional dual-beam cytometers and sorters (7,12). Chromosomes isolated from the humadhamster line (retaining human chromosomes 21, 8, and 4) were measured in the same way.…”

Section: System Resolutionsupporting

confidence: 63%

The LLNL high‐speed sorter: Design features, operational characteristics, and biological utility

et al. 1985

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This report describes a dual-beam, highspeed sorter (Hiss) with a droplet production rate of 220,000 s-l now in routine use at the Lawrence Livermore National Laboratory. The system can process and sort objects at rates in excess of 20,000 s-'. We report here on Key terms: High-speed sorting, dual-beam the development of HISS, describe its opera-flow cytometry, chromosome sorting, cell tional characteristics, and evaluate its utility sorting.for analysis and purification of human chromosomes, human erythrocytes, and living Chinese hamster ovary cells.The flow cytometer/cell sorter has become an important tool for the biomedical sciences, allowing identification and purification of subpopulations of cells and organelles with unequalled accuracy. During the last 15 yr cell sorters have been applied to purification of a broad range of cell and organelle types: for example, leukocyte and bone marrow subpopulations distinguished by their differential affinity for antibodies (8); human chromosomes distinguished by their differential uptake of one or more DNA-specific dyes (2); rare mutant erythrocytes and other peripheral blood cells distinguished by their altered affinity for antibodies against variant proteins (1); and malignant cells distinguished by their altered ability to scatter light and bind DNAspecific stains andor antibodies (11). Uses for sorted objects include simple visual study, biochemical analysis, molecular cloning, and functional assessment. Most commercially available cell sorters can scan several thousand cells per second, selecting from these the cells to be purified. Unfortunately, this processing rate is not adequate for many studies. For example, by processing objects at 2,000 s-', over 120 h of sorting time would be required to purify 1 pg of DNA from the human Y chromosome (3) and almost 140 h of sorting time would be required to sort 100 mutant erythrocytes from a population in which the mutant cell frequency wasWe have completed the development of, and put into routine operation, a dual-beam high-speed sorter (HISS) that reduces many of the limitations imposed by the relatively slow processing speed of conventional sorters. This system operates over ten times faster than conventional systems and is capable of processing and sorting cells at rates well in excess of 20,000 s-'.Most dual-beam flow cytometerhell sorters, including HiSS, operate according to the same principles (4,5,14,17). Briefly, cells or organelles in monodisperse suspension and stained with two or more fluorescent dyes are carried in single file in a fast-moving liquid stream sequentially through two tightly focused laser beams whose wavelengths are adjusted to excite the fluorescent dyes. The fluorescence andor light scatter produced during each laser beam crossing is collected by a n optical system and projected through spectral filters onto photodetectors. These detectors and their associated electronics convert the fluorescence andor scattered light to electrical pulses whose amplitudes are 'Work performed under the ...

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Section: System Resolutionsupporting

confidence: 63%

The LLNL high‐speed sorter: Design features, operational characteristics, and biological utility

et al. 1985

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“…The DNA of the human Y has been estimated to represent about 1% of the DNA of the genome of cells of normal 46,XY karyotype (Gray et al 1979) and of cells of the Chinese hamster x human hybrid line used (Cremer et al 1982). In the case of a random distribution of the silver grains, one should expect that only about 1% of the total number of silver grains should be located on the Y chromosome.…”

Section: Resultsmentioning

confidence: 99%

“…For calculation of significance, the relative DNA content for X and Y chromosome (DNA content of X(Y)/DNA content of all chromosomes of the complement) was obtained as given in Material and methods. In case of hybridization to human cells the values of Gray et al (1979) were used. For the CH-Y-VII cells, the relative DNA content of the Y was established by dual beam flow cytometry (Cremer et al 1982 Table 1).…”

Section: Resultsmentioning

confidence: 99%

Chromosome assignment of two cloned DNA probes hybridizing predominantly to human sex chromosomes

Rappold

Cremer

et al. 1984

Hum Genet

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Summary. In situ hybridization experiments were carried out with two clones, YACG35 and 2.8, which had been selected from two genomic libraries strongly enriched for the human Y chromosome. Besides the human Y chromosome, both sequences strongly hybridized to the human X chromosome, with few minor binding sites on autosomes. In particular, on the X chromosome DNA from clone YACG35 hybridized to the centromeric region and the distal part of the short arm (Xp2.2). On the Y chromosome, the sequence was assigned to one site situated in the border region between Yql.land Yql.2. DNA from clone 2.8 also hybridized to the centromeric region of the X and the distal part of the short arm (Xp2.2). On the Y, however, two binding sites were observed (Ypl.1 and Yql.2). The findings indicate that sex chromosomal sequences may be localized in homologous regions (as suggested from meiotic pairing) but also at ectopic sites.

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“…Key terms: Dual beam flow cytometry, netropsin, distamycin A counterstaining High-resolution flow cytometric chromosome analysis and sorting can be performed using both single-and dual-laser techniques (8) and combined with recombinant DNA technology to obtain chromosome-enriched phage libraries (5,(9)(10)(11)(12)18). Roughly fifteen human chromosome fluorescence populations can be identified using single-laser or univariate chromosome analysis (1) including the Y chromosome (7), although several cell lines must be screened in order to identify a Y chromosome with a distinguishable staining pattern (7).…”

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confidence: 99%

Identification of inverted duplicated #15 chromosomes using bivariate flow cytometric analysis

et al. 1985

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A dual laser FACS IV cell sorter has been used to obtain bivariate flow histograms of human metaphase chromosomes stained with the DNA-specific dyes, 33258 Hoechst and chromomycin A3. Approximately twenty distinct chromosomal fluorescence populations can be resolved using this double staining technique and the flow cytometer which has been modified only by the substitution of a specially designed air-spaced achromat for the standard focusing lens.Metaphase chromosomes from two different cell lines bearing inverted duplicated #15 autosomes have been subjected to bivariate chromosome analysis. In both cases, the inverted duplicated #15 chromosomes have been identified in the bivariate flow histogram. This identification was supported by experiments in which doubly stained chromosomes were counterstained with either netropsin or distamycin A, resulting in a relative increase in the 33258 Hoechst fluorescence intensity of the structurally abnormal #15 chromosomes, compared with the other chromosomes, as predicted by cytological studies.The possibility of identifying and separating small abnormal autosomes using commercially available instrumentation should facilitate the use of recombinant DNA techniques for the construction of libraries which are highly enriched for DNA sequences from limited autosomal subregions important in the study of chromosomal abnormalities such as deletions, translocations and inversion duplications.

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High resolution chromosome analysis: one and two parameter flow cytometry

Cited by 125 publications

References 23 publications

The LLNL high‐speed sorter: Design features, operational characteristics, and biological utility

The LLNL high‐speed sorter: Design features, operational characteristics, and biological utility

Chromosome assignment of two cloned DNA probes hybridizing predominantly to human sex chromosomes

Identification of inverted duplicated #15 chromosomes using bivariate flow cytometric analysis

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