Screen Quest Calbryte 590 免丙磺舒和免洗钙检测试剂盒
| Ex (nm) | 581 | Em (nm) | 593 |
| 分子量 | - | 溶剂 | - |
| 存储条件 | 在零下15度以下保存, 避免光照 |
钙流动测定是药物发现中筛选G蛋白偶联受体（GPCRs）的首选方法。Screen Quest Calbryte-590 无需丙磺舒（Probenecid）和洗涤的钙测定试剂盒提供了基于红色荧光的测定法，用于检测细胞内钙动员。表达具有钙信号的目标GPCR的细胞预先加载我们的专有Calbryte-590 AM，这种化合物可以穿透细胞膜。Calbryte-590 AM是用于高通量筛选（HTS）最亮的钙指示剂。一旦进入细胞，Calbryte-590 AM的脂溶性封闭基团会被非特异性的细胞酯酶切割，生成带负电荷的荧光染料，并在与钙结合时增强其荧光。刺激细胞时，筛选化合物通过受体信号释放细胞内钙，从而大大提高Calbryte-590 AM的荧光。其优异的细胞保持能力、高灵敏度和与钙结合时可增加100-250倍的荧光强度，使Calbryte-590 AM成为测量细胞钙的理想指示剂。Calbryte-590 AM是唯一无需丙磺舒（Probenecid）来提高细胞保持能力的红色钙染料。该Screen Quest Calbryte-590 无需丙磺舒和洗涤的钙测定试剂盒提供了优化的测定方法，用于检测具有难以培养的细胞系的GPCR和钙通道。与基于绿色荧光的钙测定法相比，该试剂盒受有色化合物的干扰较小。同时，它还与GFP细胞系兼容，适用于高内涵分析。该测定可以方便地在96孔或384孔微孔板格式中进行，并且易于适应自动化操作。
适用仪器
| 荧光酶标仪 | |
| Ex: | 540 nm |
| Em： | 590 nm |
| Cutoff： | 570 nm |
| 推荐孔板: | 黑色透明底板 |
| 读取模式: | 底部读取/分液处理 |
| 其他可用仪器 | |
| ArrayScan, FDSS, FlexStation, IN Cell Analyzer, NOVOStar, ViewLux |
样品实验方案
简要概述
- 在生长培养基中准备细胞
- 添加Calbryte 590 AM染料加载溶液（对于96孔板为100 µL /孔，对于384孔板为25 µL /孔）
- 在室温或37°C下孵育45-60分钟
- 在Ex / Em = 540/590 nm处检测荧光
溶液配制
储备溶液配制
1. Calbryte 590 AM储备溶液：向Calbryte 590 AM（组分A）的小瓶中加入20 µL（#36200）或200 µL（#36201和＃36202）DMSO。 注意：20 µL Calbryte 590 AM储备溶液足以装一板。 未用完的Calbryte 590 AM储备溶液可以等分分装，并在<-20℃下保存。 注意：避光，并避免重复的冻融循环。
2.分析缓冲液（1X）：将9 mL的HHBS（组分C，试剂盒#36202中未包括）与1 mL的10XPluronic®F127 Plus（10X）（组分B）充分混合。
工作溶液配制
Calbryte 590 AM工作溶液：将20 µL Calbryte 590 AM储备溶液添加到10 mL的测定缓冲液（1X）中，并充分混合。 注意：该工作溶液在室温下至少可稳定2小时。 注意：10 mL染料加载溶液足以用于一块96孔板。
实验步骤
1.将100 µL /孔（96孔板）或25 µL /孔（384孔板）的Calbryte 590 AM染料加载溶液添加到细胞板中。
2.在细胞培养箱中将染料加载板孵育60分钟，然后在室温下将板再孵育15-30分钟。 注意：如果测定需要37°C，请立即进行实验，而无需进一步室温孵育。 如果细胞在室温下能长时间正常工作，则在室温下孵育细胞板1小时（建议孵育时间不超过2小时）。
3.用HHBS或所需的缓冲液准备复合板。
4.通过检测Ex / Em = 540/590 nm的荧光强度。
图示
 图1.图表说明了信噪比（SNR）x 100％。 使用Screen Quest Calbryte 590无丙磺舒和无洗涤钙测定试剂盒测量CHO-K1细胞中的ATP剂量反应。 将CHO-K1细胞以50,000个细胞/ 100 µL /孔在96孔黑色板上接种过夜。 加入100 µL的染料加载溶液，在37°C下孵育45分钟，在室温下孵育15分钟。 FlexStation 3添加了ATP（50 µL /孔）以达到指示的浓度。 |
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