该试剂盒有多种参数可用于监控细胞活力。试剂盒中使用的专有的紫色激光激发荧光染料是一种疏水性化合物，可轻松渗透完整的活细胞，并在进入活细胞后获得增强的荧光。细胞内酯酶对非荧光底物的水解产生了强烈的蓝色荧光产物，该产物很好地保留在细胞质中。通过非荧光底物的酯酶水解产生的蓝色荧光团具有荧光素的光谱特性。当在405 nm激发时，荧光团在〜450 nm处发出强烈的蓝色荧光。该试剂盒提供了所有必需成分，并具有用于荧光微孔板检测的优化的细胞标记方案。此Cell Meter 细胞活力分析试剂盒为荧光流式细胞术，微孔板和细胞功能的显微镜研究提供了标记细胞的有效工具。它可用于多种研究，包括细胞粘附，趋化性，多药耐药性，细胞生存力，细胞凋亡和细胞毒性。该试剂盒适用于增殖和非增殖细胞。

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适用仪器

样品实验方案

简要概述

1. 用测试化合物准备细胞
2. 加入相同体积的CytoCalcein™Violet 450，AM工作溶液（100 µL /孔/ 96孔板或25 µL /孔/ 384孔板）
3. 在室温或37°C下孵育1小时
4. 监测Ex / Em = 405/460 nm（截止= 435 nm）的荧光强度（底部读取模式）

溶液配制

储备溶液配制

CytoCalcein™Violet 450，AM储备溶液：

将20 µL DMSO（组分B）添加到CytoCalcein™Violet 450，AM（组分A）的小瓶中，并充分混合以制成CytoCalcein™Violet 450，AM储备溶液。注意：20 µL CytoCalcein™Violet 450，AM储备液足以装满一块板。避光。为了存放，请将管子紧紧密封。

工作溶液配制

将CytoCalcein™Violet 450，AM储备溶液的全部内容物（20 µL）加入10 mL的测定缓冲液（组分C）中，并充分混合以制成CytoCalcein™Violet 450，AM工作溶液。该CytoCalcein™Violet 450，AM工作溶液在室温下至少可稳定2小时。注意：如果诸如CHO细胞之类的细胞含有能促进荧光染料随时间渗漏的有机阴离子转运蛋白，则应制备丙磺舒储备溶液并以最终孔内工作浓度范围为1至1的浓度添加到上样缓冲液中。 2.5毫米 分装并保存未使用的丙磺舒储备溶液于≤-20°C。

操作步骤

1. 根据需要用测试化合物处理细胞。注意：添加化合物之前不必洗涤细胞。但是，如果测试的化合物对血清敏感，则可以在添加化合物之前将生长培养基和血清因子吸掉。加入100 µL /孔（96孔板）和25 µL /孔（384孔板）的1X Hank盐溶液和20 mM Hepes缓冲液（HHBS）或抽吸后选择的缓冲液。或者，细胞可以在无血清培养基中生长。
2. 加入100 µL /孔（96孔板）或25 µL /孔（384孔板）的CytoCalcein™Violet 450，AM工作溶液。
3. 在避光的条件下，于室温或37°C下孵育平板1小时。（孵育时间可能是15分钟到过夜。孵育时间少于4小时，我们获得了最佳结果）。 注意：适当的孵育时间取决于所用的单个细胞类型和细胞浓度。优化每个实验的孵育时间。 注意： 加载后请勿洗涤细胞。 注意： 对于非贴壁细胞，建议在孵育后以800 rpm的速度离心细胞板2分钟，然后制动。
4. 使用荧光板读数器（底部读取模式）在Ex / Em = 405/460 nm（截止= 435 nm）下监控荧光强度。

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