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Qualitative and quantitative analysis of the immune synapse in the human system using imaging flow cytometry

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins towards the immune synapse to assure a stable binding and signal exchange. Classical confocal, TIRF, or super-resolution microscopy have been used to s...

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Bibliographic Details
Main Authors: Wabnitz, Guido H. (Author) , Kirchgessner, Henning (Author) , Samstag, Yvonne (Author)
Format: Article (Journal) Video
Language:English
Published: January 7, 2019
In: JoVE. Video journal
Year: 2019, Issue: 143, Pages: ?
ISSN:1940-087X
DOI:10.3791/55345
Subjects:
Film
Online Access:Verlag, lizenzpflichtig, Volltext: https://doi.org/10.3791/55345
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Author Notes:Guido Wabnitz, Henning Kirchgessner, Yvonne Samstag

MARC

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520 |a The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins towards the immune synapse to assure a stable binding and signal exchange. Classical confocal, TIRF, or super-resolution microscopy have been used to study the immune synapse. Since these methods require manual image acquisition and time-consuming quantification, the imaging of rare events is challenging. Here, we describe a workflow that enables the morphological analysis of tens of thousands of cells. Immune synapses are induced between primary human T cells in pan-leukocyte preparations and Staphylococcus aureus enterotoxin B (SEB)-loaded Raji cells as APCs. Image acquisition is performed with imaging flow cytometry, also called In-Flow microscopy, which combines features of a flow cytometer and a fluorescence microscope. A complete gating strategy for identifying T cell/APC couples and analyzing the immune synapses is provided. As this workflow allows the analysis of immune synapses in unpurified pan-leukocyte preparations and hence requires only a small volume of blood (i.e., 1 mL), it can be applied to samples from patients. Importantly, several samples can be prepared, measured, and analyzed in parallel. 
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