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

Details

• Alternative Title:
  Curr Protoc Stem Cell Biol
• Personal Author:
  Luecke, Linda Berg ; Waas, Matthew ; Gundry, Rebekah L.
• Description:
  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.
• Subjects:
  Cell Culture Techniques Cell Differentiation Cell Line Flow Cytometry Humans Myocytes, Cardiac Pluripotent Stem Cells
• Source:
  Curr Protoc Stem Cell Biol. 50(1):e94
• Pubmed ID:
  31479597
• Pubmed Central ID:
  PMC6727984
• Document Type:
  Journal Article
• Funding:
  TL1 TR001437/TR/NCATS NIH HHSUnited States/ ; R01 HL126785/HL/NHLBI NIH HHSUnited States/ ; T32 GM080202/GM/NIGMS NIH HHSUnited States/ ; R01 HL134010/HL/NHLBI NIH HHSUnited States/ ; F31 HL140914/HL/NHLBI NIH HHSUnited States/ ; U01 CE002944/CE/NCIPC CDC HHSUnited States/ ; UL1 TR001436/TR/NCATS NIH HHSUnited States/ ; U01 NS088034/NS/NINDS NIH HHSUnited States/
• Volume:
  50
• Issue:
  1
• Collection(s):
  CDC Public Access
• Main Document Checksum:
  urn:sha256:81ede171158b1e99f0fb6ac91c8ae33844c4f3e008e1becc871800a6ebcbaf3d
• Download URL:
  https://stacks.cdc.gov/view/cdc/119164/cdc_119164_DS1.pdf
• File Type:
  [PDF - 1.26 MB ]