PubMed · 9702764
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M Schindler. 1998. Select, microdissect, and eject.. https://doi.org/10.1038/nbt0898-719
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BACKGROUND: Successful DNA typing after rape is limited when only a few sperm and numerous vaginal cells are recovered from a swab, resulting in an extremely unfavorable ratio of male to female DNA. The goal of this study was to develop a protocol involving sperm cell sorting with flow cytometry based on differences in ploidy, major histocompatibility (MHC) class I, CD45 and cytokeratin expression. METHODS: Vaginal lavages were mixed with serially diluted ejaculate. After immunostaining and stoichiometric nuclear staining, spermatocytes were isolated by fluorescence-activated cell sorting. All sorted cells were used for DNA extraction and subsequent quantitative fluorescent multiplex polymerase chain reaction. The preferential lysis was performed for comparison. RESULTS: The sorting procedure was superior to the preferential lysis method within all tested dilutions. One documented case of rape was examined with both procedures and only after cell sorting with flow cytometry was the male DNA identified. CONCLUSIONS: We were able to show that separation of sperm and vaginal cells using cell sorting with flow cytometry may be crucial when there is only a few sperm detectable after rape.
Magnetic separation is an emerging technology using magnetism, sometimes in combination with conventional separation or identification methods, to purify cells, cell organelles and biologically active compounds (nucleic acids, proteins, xenobiotics) directly from crude samples. Several magnetic separation procedures have been developed to isolate target cells specifically. The purpose of this short review is to summarize various methodologies, strategies and materials which can be employed for the selection and separation of target cells with the help of magnetic field and thus to help the novices in this field to be able to orient themselves in vast amount of literature available. Immunomagnetic separations employing specific antibodies to label the target cells represent the most often used approach and are discussed in detail.
OBJECTIVE: To characterize the expression and regulation of conjunctival mast cell surface receptors important in allergic inflammation. METHODS: Mast cells were isolated from human conjunctival tissues of cadavers. Mast cell surface markers were identified using flow cytometry with antibodies to IgE, Fc epsilonRI, c-kit, and intercellular adhesion molecule-1 (ICAM-1). We evaluated the effect of 24-hour tumor necrosis factor alpha (TNF-alpha) or interleukin 4 (IL-4) incubation on the expression of mast cell c-kit, ICAM-1, and surface-bound IgE. RESULTS: Staining of mast cells (c-kit and/or tryptase positive) yielded positive results for all of the variables measured. The intensity of mast cell c-kit staining increased with TNF-alpha incubation, but decreased below that of unstimulated mast cells when incubated with IL-4. Anti-ICAM-1 and anti-IgE staining were increased over that of unstimulated cells when incubated with TNF-alpha or IL-4. CONCLUSIONS: In this model, TNF-alpha up-regulates mast cell surface receptors and cell-bound IgE. Interleukin 4 up-regulates mast cell ICAM-1 and cell-bound IgE, but down-regulates c-kit. CLINICAL RELEVANCE: Conjunctival mast cells play a critical role in the pathogenesis of atopic ocular disease. Characterization of the expression and regulation of mast cell surface receptors is important to the development of potential novel treatments for ocular inflammation.