@article{95a06d0ac77944989266c5bbdeff7a7c,
title = "Reliable Protocols for Flow Cytometry Analysis of Intracellular Proteins in Pluripotent Stem Cell Derivatives: A Fit-For-Purpose Approach",
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.",
keywords = "flow cytometry, hPSC-CM, heterogeneity, intracellular proteins, quality control",
author = "{Berg Luecke}, Linda and Matthew Waas and Gundry, {Rebekah L.}",
note = "Funding Information: This work was supported by the National Institutes of Health [R01‐HL126785 and R01‐HL134010] to R.L.G.; F31‐HL140914 to M.W.; and support received by the National Center for Advancing Translational Sciences, National Institutes of Health, through grant numbers UL1TR001436 and TL1TR001437 to L.B.L. L.B.L. is a member of the MCW‐MSTP, which is partially supported by a T32 grant from NIGMS, GM080202. Funding sources were not involved in study design, data collection, interpretation, analysis, or publication. Flow cytometry analyses were performed using instrumentation in the Blood Center of Wisconsin Flow Cytometry Core. Funding Information: This work was supported by the National Institutes of Health [R01-HL126785 and R01-HL134010] to R.L.G.; F31-HL140914 to M.W.; and support received by the National Center for Advancing Translational Sciences, National Institutes of Health, through grant numbers UL1TR001436 and TL1TR001437 to L.B.L. L.B.L. is a member of the MCW-MSTP, which is partially supported by a T32 grant from NIGMS, GM080202. Funding sources were not involved in study design, data collection, interpretation, analysis, or publication. Flow cytometry analyses were performed using instrumentation in the Blood Center of Wisconsin Flow Cytometry Core. Publisher Copyright: {\textcopyright} 2019 John Wiley & Sons, Inc.",
year = "2019",
month = sep,
doi = "10.1002/cpsc.94",
language = "English (US)",
volume = "50",
journal = "Current Protocols in Stem Cell Biology",
issn = "1941-7322",
publisher = "John Wiley and Sons Inc.",
number = "1",
}