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

Autor: Berg Luecke L; Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin., Waas M; Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin., Gundry RL; Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin.; Center for Biomedical Mass Spectrometry Research, Medical College of Wisconsin, Milwaukee, Wisconsin.
Jazyk: angličtina
Zdroj: Current protocols in stem cell biology [Curr Protoc Stem Cell Biol] 2019 Sep; Vol. 50 (1), pp. e94.
DOI: 10.1002/cpsc.94
Abstrakt: 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.
(© 2019 John Wiley & Sons, Inc.)
Databáze: MEDLINE
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