A novel method that simultaneously monitors the low fluorescence intensity of Handbook of Immunohistochemistry and in situ Hybridization of Human Carcinomas, Volume 3Handbook of Immunohistochemistry and in Situ Hybridization of Human CarcinomasEncyclopedia of Food Sciences and Nutrition (Second Edition)From the onset of the use of flow cytometry for sex separation, using By staining intact cells with the DNA binding, vital, fluorescent dye, Pathological Assessment of Experimental Models of Stem Cell and Other Regenerative TherapiesStem Cell and Gene Therapy for Cardiovascular DiseaseScienceDirect ® is a registered trademark of Elsevier B.V. Clipboard, Search History, and several other advanced features are temporarily unavailable. Expression of a Hoechst 33342 efflux phenomenon and common characteristics of pluripotent stem cells in a side population of amniotic fluid cells. This site needs JavaScript to work properly. HighWire Within each population, emission is still proportional to DNA content; thus, it is possible to define subsets in different phases of the cell cycle.Observe the cells directly under an inverted fluorescence microscope.If desired, fix the cells after vital staining of transfected cells for further detailed analysis of morphology: These include We use cookies to help provide and enhance our service and tailor content and ads. We outline a simple approach involving instrument setup and calibration for the analysis of Hoechst dye 33342-loading in human cell lines for exploring heterogeneity in dye efflux efficiency and the status of side population (SP) A549 lung cancer cells. Wash the cells with PBS (this step can be omitted), and then fix the cells with 4% (w/v) paraformaldehyde or 1% (v/v) glutaraldehyde for 5 min at room temperature.Samples are mounted with PermaFluor aqueous mounting medium and cells are examined under a fluorescence microscope equipped with the appropriate filters.Washing should be done as gently as possible to minimize the loss of dead cells.The SP assay has also been applied to human hematopoietic tissues (Configure the lasers and detectors for excitation and detection of Select pinholes equal to 1 Airey unit for each optical path.Place the slide on the microscope and focus on a fibroblast centrosome.Set the digital zoom at 2X and the line averaging at 4.Set laser power and detection parameters so that the resulting signal is saturated or nearly saturated only at the centrosome. Hoechst 33342 is a bis-benzimide derivative that binds to AT-rich sequences in the minor grove of double-stranded DNA.The advantage of Hoechst 33342 staining is the low cost of reagent and high degree of HSC enrichment, making the side population an appealing method to isolate HSC; even without additional purification, about 75% of SP cells express the cell surface markers of mouse HSC as defined by most other methods (i.e., KSL, CD34The meticulousness of Hoechst staining often varies from laboratory to laboratory, as evident by the reported discrepancies of marker expressions and function of SP and non-SP cells found among different groups, leading to the side population sometimes being considered a “controversial” marker. Copyright © 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved. The side population pattern is a result of both the nonlinear emission of Hoechst 33342 dye under UV excitation (Petersen et al., 2004) and the distinct ability of bone marrow cells to retain Hoechst dyes, and also likely the chromatin conformation within cell populations, which results in differential access of the dye to the DNA. For instance, the side population in one report was shown to fail to included all HSCs as there were functional long-term HSCs in the non-SP cells (Dual-wavelength analysis of Ho-stained murine bone marrow reveals two striking aspects: the presence of more than one GIf different bone marrow cell types possess distinct chromatin conformations, we would expect to find different subpopulations with Ho staining, provided that the dye concentration is adequate. The diameter of the circle is not critical; just make sure that it includes the entire centrosome signal and excludes any autofluorescence your sample may have.Select the area button to obtain the area of the centrosome signal inside the circle.Count the number of the nuclei in the field and enter the number in the spreadsheet.Once the measurements have been collected, you should have a spreadsheet for each tissue analyzed. HighWire Tsai YL(1), Chang YJ, Chou CY, Cheong ML, Tsai MS.

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