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Techniques for Imaging Ca2+ Signaling in Human Sperm
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June 16, 2010
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Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.
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Fluorescence MicroscopyCa2+ SignalingHuman SpermFluorescent DyesLoading MethodPhysiological StimulationCapacitating ConditionsAM EsterCytoplasmVisible Wavelength DyesRatiometric RecordingAdvantages And DisadvantagesImaging ChamberPoly-D-lysine Coated CoverslipPerfusion ApparatusTime-lapse AcquisitionRegions Of InterestData Normalization
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Nash, K., Lefievre, L., Peralta-Arias, R., Morris, J., Morales-Garcia, A., Connolly, T., Costello, S., Kirkman-Brown, J. C., Publicover, S. J. Techniques for Imaging Ca2+ Signaling in Human Sperm. J. Vis. Exp. (40), e1996, doi:10.3791/1996 (2010).
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