Protocol for Barcoding T Cells Combined with Timed Stimulations

STAR Protoc. 2020 Jul 22;1(2):100067. doi: 10.1016/j.xpro.2020.100067. eCollection 2020 Sep 18.

Abstract

Stimulation of naive T lymphocytes via the T cell receptor (TCR) induces distinct phosphorylation patterns that can be used to explore various signaling pathways within the cell. This protocol can be used to characterize different perturbations to the signaling pathways and the variations in time of stimulation. Here, we provide a method of barcoding and consolidating a maximum of 24 different sample conditions using two florescent dyes. This single sample for phospho-staining and flow cytometry saves time and reagents. For complete details on the use and execution of this protocol, please refer to Krutzik and Nolan (2006), Krutzik et al. (2012), Vercoulen et al. (2017), Ksionda et al. (2018), and Myers et al. (2019).

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cells, Cultured
  • Flow Cytometry / methods*
  • Fluorescent Dyes* / chemistry
  • Fluorescent Dyes* / metabolism
  • Mice
  • Molecular Probe Techniques*
  • Phosphorylation
  • Receptors, Antigen, T-Cell / chemistry
  • Receptors, Antigen, T-Cell / metabolism
  • Signal Transduction
  • T-Lymphocytes* / chemistry
  • T-Lymphocytes* / cytology
  • T-Lymphocytes* / metabolism
  • Time Factors

Substances

  • Fluorescent Dyes
  • Receptors, Antigen, T-Cell