Micro-2D Cell Culture for cAMP Measurements Using FRET Reporters in Human iPSC-Derived Cardiomyocytes.

Abstract

In the last years human induced pluripotent stem cell-derived cardiomyocytes (hIPS-CMs) have emerged as a promising alternative to rodent-derived cardiomyocytes. However, as the differentiation process is lengthy and commercially available cells are expensive, the cell number is limited. Here we provide detailed information on how to scale down 2D cell cultures of hIPS-CMs for the purpose of cAMP FRET measurements, thereby extending the number of possible experiments by more than tenfold. Crucial factors like cell density or cell number to culturing media volume can be maintained exactly as under normal culturing conditions and existing equipment does not need to be modified.The chapter covers the preparation of downscaled cell culture vessels, coating and seeding procedures, transduction or transfection of the cells with a genetically encoded cAMP FRET sensor, performing real-time cAMP FRET measurements with this sensor and the analysis of generated imaging data. Numbers for seeding areas, seeding densities, coating volumes and concentrations, media volumes, and concentrations of reagents are given as guidelines.