Institute of Pharmacology and Toxicology, University of Würzburg, Würzburg, Germany.
Nat Protoc. 2011 Apr;6(4):427-38. doi: 10.1038/nprot.2010.198. Epub 2011 Mar 10.
Real-time measurements of second messengers in living cells, such as cAMP, are usually performed by ratiometric fluorescence resonance energy transfer (FRET) imaging. However, correct calibration of FRET ratios, accurate calculations of absolute cAMP levels and actual permeabilities of different cAMP analogs have been challenging. Here we present a protocol that allows precise measurements of cAMP concentrations and kinetics by expressing FRET-based cAMP sensors in cells and modulating them with an inhibitor of adenylyl cyclase activity and a cell-permeable cAMP analog that fully inhibits and activates the sensors, respectively. Using this protocol, we observed different basal cAMP levels in primary mouse cardiomyocytes, thyroid cells and in 293A cells. The protocol can be generally applied for calibration of second messenger or metabolite concentrations measured by FRET, and for studying kinetics and pharmacological properties of their membrane-permeable analogs. The complete procedure, including cell preparation and FRET measurements, takes 3-6 d.
实时测量细胞内的第二信使，如 cAMP，通常通过比率荧光共振能量转移（FRET）成像来实现。然而，FRET 比率的正确校准、绝对 cAMP 水平的精确计算以及不同 cAMP 类似物的实际渗透率一直是具有挑战性的。在这里，我们提出了一种方案，通过在细胞中表达基于 FRET 的 cAMP 传感器，并分别用腺苷酸环化酶活性抑制剂和一种细胞通透的 cAMP 类似物来调节它们，从而可以精确测量 cAMP 浓度和动力学。使用该方案，我们观察到原代小鼠心肌细胞、甲状腺细胞和 293A 细胞中的不同基础 cAMP 水平。该方案通常可用于通过 FRET 测量的第二信使或代谢物浓度的校准，以及研究其膜通透类似物的动力学和药理学特性。完整的程序，包括细胞准备和 FRET 测量，需要 3-6 天。
