适用仪器

样品实验方案

简要概述

准备含测试化合物的细胞溶液
取出介质
加入CytoCalcein™NIR工作溶液（100 µL /孔/ 96孔板或25 µL /孔/ 384孔板）
在室温或37°C下孵育1小时
在Ex / Em = 635/670 nm（截止= 665 nm）时读取荧光强度（底部读取模式）

溶液配制

1.储备溶液配制

除非另有说明，否则所有未使用的储备溶液应分成一次性等分试样，并在制备后储存在-20°C。 避免反复冻融循环。
CytoCalcein™NIR储备溶液：
将20 µL DMSO（组分B）添加到CytoCalcein™NIR（组分A）的小瓶中，并充分混合以制成CytoCalcein™NIR储备液。 注意：20 µL CytoCalcein™NIR储备液足以装满一块板。 避光。 为了存放，请将管子密封。

2.工作溶液配制

将20 µL CytoCalcein™NIR储备溶液添加到瓶中的测定缓冲液（10 mL，组分C）中并充分混合以制成CytoCalcein™NIR工作溶液。 注意：此CytoCalcein™NIR工作溶液适用于一块细胞板，在室温下至少可稳定2小时。有关细胞样品制备的指南，请点击查看。

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.从细胞中除去培养基。注意：加载CytoCalcein™NIR工作溶液之前，必须除去培养基。

3.加入100 µL /孔（96孔板）或25 µL /孔（384孔板）的CytoCalcein™NIR工作溶液。

4.在室温或37°C下避光放置1小时。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。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。

5.使用荧光酶标仪（底部读取模式）在Ex / Em = 635/670 nm（截止= 665 nm）下监控荧光强度。

参考文献

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