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  • 1
    ISSN: 1040-452X
    Keywords: Heterogeneity of human sperm ; Condensation ; Chromatin structure ; Laser scanning cytometry ; Flow cytometry ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Flow cytometric studies of human sperm from fertile men display a constant and characteristic bimodal nonartifactual DNA pattern confirming the existence of two distinct populations. The main population is represented by a peak followed by a shoulder (“marginal population”). The appearance of this marginal population fluctuates with either freezing and thawing or with Percoll gradient centrifugation.We have analyzed both the main and marginal sperm populations by flow cytometry after cell sorting, laser scanning cytometry, light microscopic evaluation, and their sensitivity to DNase digestion.We have observed that the marginal population detected in fertile men represents a sperm group altered in the nuclear condensation, yielding unstable chromatin which appears more stainable with propidium iodide. © 1994 Wiley-Liss, Inc.
    Additional Material: 2 Ill.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1572-9931
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract To purify mouse Y chromosomes by flow cytometry, a male cell line containing the Robertsonian translocation Rb(9.19)163H has been established by SV40 transformation. Flow karyotypes obtained from these cells exhibit a well-isolated peak of fluorescence corresponding to the single Y chromosome, clearly distinct from that of chromosome 19. From this peak, 650,000 chromosomes were sorted, and two restriction fragment libraries were constructed from the DNA of the sorted chromosomes. The characterization of several Y-specific fragments has shown that the Y DNA was enriched at least 36-fold. Furthermore, given that there are likely homologies between the X and Y chromosomes, we can assume that this calculated value of the purification factor is an underestimation and that the Y DNA was more highly purified by flow sorting.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1573-0778
    Keywords: cell viability ; flow cytometry ; light absorption ; light scattering ; exclusion dyes ; supravital dyes ; fluorescence
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Flow cytometry offers the possibility to simultaneously analyze, on a cell by cell basis, different parameters related to cell viability i.e. cell size, morphology and incorporation of dyes. Different types of analysis: light absorption of unstained/stained cells, forward angle light scattering (FALS), right angle light scattering (RALS) or both, cell fluorescence based on dye retention or dye exclusion (due to erythrosin B, ethidium bromide, fluorescein diacetate, rhodamine 123) were tested and compared, with the classical Trypan blue exclusion test, for their effectiveness in the determination of cell viability. Two types of cells in monolayer cultures (L929, SIRC) and a freshly isolated suspension of mouse splenocytes were used. For each dye, the optimal dose, incubation time and conditions for analysis were determined. Viability indications by different techniques for the three type of cell line and their reliability as compared with Trypan blue were analyzed.
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 1040-452X
    Keywords: Flow cytometry ; Sperm sorting ; DNA staining ; Bovine Y specific probe ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Flow cytometry is a potential method for the separation of X and Y bearing spermatozoa, on the basis of their relative DNA content evaluated by the fluorescence emission intensity due to specific fluorochrome DNA staining. However, spermatozoa DNA is highly condensed and nuclei exhibit flat non spherical shape, which can produce artefacts impeding accurate analysis. In order to avoid these limitations, decondensation of DNA performed by enzymatic treatment and a modification of the flow cytometer that orients the spermatozoa relative to the laser beam are generally used. In this work, we describe alternative methods and materials for selection of (1) decondensed and thus dead spermatozoa without orientation, sorted on the basis of only the 10% spermatozoa containing the least DNA (expected Y) and the 10% spermatozoa containing the more DNA (expected X), or (2) native spermatozoa homogeneously oriented using a simultaneous measurement of Axial light loss (extinction) and Forward angle light scatter. For testing enrichment of each selected fraction we have worked out a molecular hybridization procedure using X and Y specific DNA probes. We analyse and sort bull spermatozoa on these basis: the purity obtained for these fractions is 80% without orientation after enzymatic treatment, and 70% on live spermatozoa “optically” oriented.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
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