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)以达到最终指示的浓度。
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