| Lysosomal Stability Assay
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Author:
Date:
2014-06-20
[Abstract] This assay makes use of the dye Acridine Orange (AO) to determine the stability of lysosomes in living cells upon exposure to a confocal microscope laser.
AO is a lipophilic amine that readily diffuses into cells (Figure 1). Inside the cell it enters the acidic lysosomal compartment where it is protonated and sequestered, shifting its emission spectrum towards a longer wavelength (i.e. red). Once inside the lysosomes, the metachromatic AO sensitizes the lysosomal membrane to photo-oxidation by blue light (Brunk et al., 1997). Upon light-induced loss of the lysosomal pH gradient and subsequent leakage of AO into the cytosol, the emission spectrum of AO shifts from red to green (Figure 2). Hence, loss of lysosomal integrity can be measured as a ‘loss of red ...
[摘要] 该测定利用染料吖啶橙（AO）确定在暴露于共焦显微镜激光器时活细胞中溶酶体的稳定性。
图1。吖啶橙
AO是一种易于扩散入细胞的亲脂胺（图1）。在细胞内部，它进入酸性溶酶体区室，在那里它被质子化和螯合，将其发射光谱移向更长的波长（即，红色）。一旦在溶酶体内，异染色质AO使溶酶体膜通过蓝光的光氧化敏感（Brunk等人，1997）。在光诱导的溶酶体pH梯度的损失和随后的AO向胞质溶胶中的泄漏后，AO的发射光谱从红色变为绿色（图2）。因此，溶酶体完整性的损失可以被测量为"红点的丢失"或作为绿色荧光的定量升高（Petersen等人，2010; Kirkegaard等人，，2010; Petersen ，2013）。
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| Three-dimensional Invasion Assay
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Author:
Date:
2013-09-05
[Abstract] The invasive ability of cancer cells is a crucial function for cancer metastasis and the surrounding microenvironment of cancer cells in living tissues is three-dimension (3D). Therefore, to establish an in vitro invasion assay in a 3D system to predict cancer invasive ability is valuable in the research for cancer metastasis. Here, we describe a 3D invasion assay for observing the morphology and comparing the invasive ability of cancer cells in artificial 3D environments (Yang et al., 2012). Collagen I gels are used to cover on the top of cancer cells attached on coverslip glass dish and medium containing FBS is added as a chemoattractant. After incubation for a suitable time, the cells are fixed and stained. The invasion index can be calculated and the morphology can ...
[摘要] 癌细胞的侵袭能力是癌症转移的关键功能，活组织中癌细胞的周围微环境是三维的（3D）。 因此，在3D系统中建立体外侵袭测定以预测癌症侵袭能力在癌症转移的研究中是有价值的。 在这里，我们描述了用于观察形态学并比较人造3D环境中癌细胞的侵袭能力的3D侵袭测定（Yang等人，2012）。 胶原I凝胶用于覆盖附着在盖玻片玻璃皿上的癌细胞的顶部，并且添加含有FBS的培养基作为化学引诱物。 在孵育合适的时间后，将细胞固定并染色。 可以计算入侵指数，并且可以用激光共聚焦显微镜成像形态
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