Reliable Protocols for Flow Cytometry Analysis of Intracellular Proteins in Pluripotent Stem Cell Derivatives: A Fit-For-Purpose Approach

Q2 Biochemistry, Genetics and Molecular Biology

Current Protocols in Stem Cell Biology Pub Date : 2019-08-02 DOI:10.1002/cpsc.94

Linda Berg Luecke, Matthew Waas, Rebekah L. Gundry

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引用次数: 6

Abstract

Human pluripotent stem cell (hPSC) derivatives are valuable for a variety of research applications and have the potential to revolutionize approaches to personalized medicine. However, differentiation efficiency varies among cell lines and protocols. Therefore, methods to reliably determine cell type identity in cultures of hPSC derivatives in a manner that is consistent among laboratories are needed. While flow cytometry is apt for routine assessment of population heterogeneity, standardized protocols are not available for most cell types. This article describes a workflow for establishing a fit-for-purpose protocol for flow cytometric analysis of hPSC derivatives. Based on the application of this workflow, a standard operating procedure (SOP) was developed for the analysis of cardiac troponin in hPSC-derived cardiomyocytes (hPSC-CM). Throughout the article, important concepts related to antibody validation and gating strategies are presented to enable users to properly validate any antibody of interest and develop a rigorous SOP for their experimental needs. © 2019 by John Wiley & Sons, Inc.

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流式细胞术分析多能干细胞衍生物中细胞内蛋白的可靠方案:一种适合目的的方法

人类多能干细胞(hPSC)衍生物在各种研究应用中都很有价值，并有可能彻底改变个性化医疗的方法。然而，分化效率因细胞系和方案而异。因此，需要可靠地确定hPSC衍生物培养中细胞类型的方法，这种方法在实验室之间是一致的。虽然流式细胞术适合于群体异质性的常规评估，但大多数细胞类型都没有标准化的方案。这篇文章描述了一个工作流建立一个适合的目的协议流式细胞分析的hPSC衍生物。在此工作流程的基础上，建立了分析人乳头瘤细胞源性心肌细胞(hpc - cm)心肌肌钙蛋白的标准操作程序(SOP)。在整篇文章中，介绍了与抗体验证和门控策略相关的重要概念，以使用户能够正确验证任何感兴趣的抗体，并为他们的实验需求制定严格的SOP。©2019 by John Wiley &儿子,Inc。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Current Protocols in Stem Cell Biology Biochemistry, Genetics and Molecular Biology-Cell Biology

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期刊介绍： Published in affiliation with the International Society for Stem Cell Research (ISSCR), Current Protocols in Stem Cell Biology (CPSC) covers the most fundamental protocols and methods in the rapidly growing field of stem cell biology. Updated monthly, CPSC will constantly evolve with thelatest developments and breakthroughs in the field. Drawing on the expertise of leading researchers from around the world, Current Protocols in Stem Cell Biology includes methods and insights that will enhance the progress of global research.