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Testing the Efficiency and Kinetics of Negative Selection Using Thymic Slices

  • Tyng-An Zhou
  • Chia-Lin Hsu
  • Ivan Lilyanov DzhagalovEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2111)

Abstract

Central tolerance is an efficient barrier to autoimmunity and negative selection of self-reactive thymocytes is one of its major manifestations. Because of its importance, negative selection has been studied extensively through numerous in vitro and in vivo approaches that have tremendously increased our understanding of the process. Recently, in situ experimental systems using thymus slices have been developed that combine some of the advantages of in vitro assays such as ease of manipulation and high throughput with the existence of three dimensional mature thymus microenvironment. These approaches offer unprecedented opportunity to study negative selection. Here, we describe how thymic slices can be used to measure the kinetics and magnitude of negative selection. Taking the OT-1/Ova system as an example, we provide detailed guidance on cutting thymic slices, labeling and overlaying thymocytes on them and reading out the extent of negative selection by flow cytometry. The system can easily be adapted to evaluate the effects of various mutations or treatments on negative selection or to study the behavior of different cells in the thymus through time-lapse imaging.

Key words

Thymus Thymocytes Negative selection Central tolerance Flow cytometry Thymic slices Vibratome OT-1 Ova 

Notes

Acknowledgments

We would like to thank Hsing-Kai Feng and Yun-Tzu Chen for help with taking the photos and carefully reviewing the manuscript. This work was supported by grants from MOST, 106-2320-B-010-026-MY3 (ILD), 107-2320-B-010-016-MY3 (ILD), 104-2628-B-010-002-MY4 (CLH), and 107-2320-B-010-020 (CLH), and a grant from the Yen Tjing Ling Medical Foundation, CI-108-5 (ILD).

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Tyng-An Zhou
    • 1
  • Chia-Lin Hsu
    • 1
  • Ivan Lilyanov Dzhagalov
    • 1
    Email author
  1. 1.Institute of Microbiology and ImmunologyNational Yang-Ming UniversityTaipeiTaiwan

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