Techniques for imaging Ca2+ signaling in human sperm.
Techniques for imaging Ca2+ signaling in human sperm.
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DOI：
10.3791/1996
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发表时间：
2010-06-16
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影响因子：
--
通讯作者：
Publicover, Stephen J
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文献类型：
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作者：
Nash, Katherine;Lefievre, Linda;Publicover, Stephen J
荧光显微镜的细胞加载荧光，钙（2+）敏感染料用于测量的空间和时间方面的钙（2+）信号在活细胞。在这里，我们描述的方法，在我们的实验室加载悬浮液的人精子与Ca（2+）报告染料和测量荧光信号在生理刺激。根据实验，通过直接上游分离运动细胞，并在获能条件下孵育0-24小时。然后将Ca（2+）报告染料的细胞渗透AM（乙酰氧基甲酯）酯形式加入细胞等分试样中，并允许1小时的时间将染料加载到细胞质中。我们使用可见波长染料来最小化对细胞的光损伤，但这意味着比率记录是不可能的。这种方法的优点和缺点进行了讨论。在加载期间，将细胞引入成像室中，并使其粘附到聚-D-赖氨酸包被的盖玻片上。在加载期结束时，通过将腔室连接到灌注装置来去除过量的染料和松散的细胞。腔室连续灌注，然后将刺激物和改性盐水添加到灌注头。实验通过延时采集荧光图像进行记录，并通过手动绘制感兴趣区域进行离线详细分析。将数据标准化为刺激前水平，使得对于每个细胞（或细胞的一部分），获得显示Ca（2+）响应为荧光变化%的图。
Fluorescence microscopy of cells loaded with fluorescent, Ca(2+)-sensitive dyes is used for measurement of spatial and temporal aspects of Ca(2+) signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca(2+)-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca(2+)-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca(2+) response as % change in fluorescence is obtained.