A flow-cytometry-based assessment of global protein synthesis in human senescent cells

STAR Protoc. 2021 Sep 14;2(3):100809. doi: 10.1016/j.xpro.2021.100809. eCollection 2021 Sep 17.

Abstract

Senescent cells constantly experience stressful conditions and restrain their protein translation to cope with it. Here, we present a detailed protocol to measure the rate of global protein synthesis using L-azidohomoalanine (L-AHA)-based click chemistry in human senescent fibroblasts. We optimized several aspects of the procedure, including senescence induction, a flow cytometry analysis of senescent cells, and the duration of L-AHA incorporation. This protocol uses senescent human fibroblasts but can be applied to other types of cells or circumstances. For complete details on the use and execution of this protocol, please refer to Lee et al. (2021).

Keywords: Cell Biology; Cell-based Assays; Flow Cytometry/Mass Cytometry; Molecular Biology; Molecular/Chemical Probes; Protein Biochemistry.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alanine / analogs & derivatives
  • Alanine / analysis
  • Alanine / metabolism
  • Animals
  • Cell Line
  • Cellular Senescence / physiology*
  • Click Chemistry
  • Fibroblasts / cytology
  • Fibroblasts / metabolism
  • Flow Cytometry / methods*
  • HEK293 Cells
  • Humans
  • Mice
  • Protein Biosynthesis / physiology*
  • Proteins / analysis*
  • Proteins / chemistry
  • Proteins / metabolism

Substances

  • Proteins
  • azidohomoalanine
  • Alanine