Super-resolution Imaging of the T cell Central Supramolecular Signaling Cluster Using Stimulated Emission Depletion Microscopy

Sin Lih Tan, Dominic Alibhai, Stephen J Cross, Harry Thompson, Christoph Wuelfing

Research output: Contribution to journalArticle (Academic Journal)peer-review

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Abstract

Supramolecular signaling assemblies are of interest for their unique signaling properties. A μm scale signaling assembly, the central supramolecular signaling cluster (cSMAC), forms at the center interface of T cells activated by antigen presenting cells (APC). The adaptor protein linker for activation of T cells (LAT) is a key cSMAC component. The cSMAC has widely been studied using total internal reflection fluorescence microscopy of CD4+ T cells activated by planar APC substitutes. Here we provide a protocol to image the cSMAC in its cellular context at the interface between a T cell and an APC. Super resolution stimulated emission depletion microscopy (STED) was utilized to determine the localization of LAT, that of its active, phosphorylated form and its entire pool. Agonist peptide-loaded APCs were incubated with TCR transgenic CD4+ T cells for 4.5 min before fixation and antibody staining. Fixed cell couples were imaged using a 100x 1.4 NA objective on a Leica SP8 AOBS confocal laser scanning microscope. LAT clustered in multiple supramolecular complexes and their number and size distributions were determined. Using this protocol, cSMAC properties in its cellular context at the interface between a T cell and an APC could be quantified.
Original languageEnglish
Article numbere3806
Number of pages12
JournalBio-protocol
Volume10
Issue number21
DOIs
Publication statusPublished - 5 Nov 2020

Bibliographical note

Acceptance date is provisional and based on date of publication.

Keywords

  • immune synapse
  • cSMAC
  • STED
  • LAT
  • T cell activation

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