Department of Biomedical Engineering, School of Engineering and Applied Science, The George Washington University, Washington, DC, USA.
Methods Mol Biol. 2022;2485:15-37. doi: 10.1007/978-1-0716-2261-2_2.
We describe a method for protein quantification and for mRNA quantification in small sample quantities of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Demonstrated here is how the capillary-based protein detection system Wes™ by ProteinSimple and the Power SYBR™ Green Cells-to-CT™ Kit by Invitrogen can be applied to individual samples in a 96-well microplate format and thus made compatible with high-throughput (HT) cardiomyocyte assays. As an example of the usage, we illustrate that Cx43 protein and GJA1 mRNA levels in hiPSC-CMs are enhanced when the optogenetic actuator, channelrodopsin-2 (ChR2), is genetically expressed in them. Instructions are presented for cell culture and lysate preparations from hiPSC-CMs, along with optimized parameter settings and experimental protocol steps. Strategies to optimize primary antibody concentrations as well as ways for signal normalization are discussed, i.e., antibody multiplexing and total protein assay. The sensitivity of both the Wes and Cells-to-CT kit enables protein and mRNA quantification in a HT format, which is important when dealing with precious small samples. In addition to being able to handle small cardiomyocyte samples, these streamlined and semi-automated processes enable quick mechanistic analysis.
我们描述了一种用于在人类诱导多能干细胞衍生的心肌细胞（hiPSC-CM）的小样本量中进行蛋白质定量和 mRNA 定量的方法。这里展示的是如何将基于毛细管的蛋白质检测系统 Wes™（由 ProteinSimple 提供）和 Invitrogen 的 Power SYBR™ Green Cells-to-CT™ Kit 应用于 96 孔微孔板格式的单个样本中，从而与高通量（HT）心肌细胞测定兼容。作为使用的一个示例，我们说明了当光遗传学激活剂通道视紫红质-2（ChR2）在 hiPSC-CM 中基因表达时，Cx43 蛋白和 GJA1 mRNA 水平会增强。提供了 hiPSC-CM 的细胞培养和裂解物制备的说明，以及优化的参数设置和实验方案步骤。讨论了优化初级抗体浓度的策略以及信号归一化的方法，即抗体多重化和总蛋白测定。Wes 和 Cells-to-CT 试剂盒的灵敏度允许在 HT 格式下进行蛋白质和 mRNA 定量，这在处理珍贵的小样本时非常重要。除了能够处理小的心肌细胞样本外，这些简化和半自动化的流程还能够实现快速的机制分析。
