Protocol to Isolate Germinal Centers by Laser Microdissection

Farbod Bahreini, Markus Niebuhr, Julia Belde, Jürgen Westermann, Kathrin Kalies*

*Corresponding author for this work

Abstract

During adaptive immune responses, germinal centers (GC) appear as transient microstructures, in which antigen-specific B and T cells interact with each other. Because only the antigen-activated B and T cells, such as Plasmablasts or follicular T helper (Tfh) cells, are present in GC, the in depth-analysis of GC is of great interest. To identify the cells that reside within GC, the majority of studies use the expression of specific surface molecules for analysis by flow cytometry. To do so, the tissue has to be disrupted for the preparation of single-cell suspensions. Thereby, the local information regarding neighborhoods of B cells and T cells and their potential interaction is lost. To study GC in vivo within their original microenvironment, we established a protocol for the isolation of GC by laser microdissection. To enable the identification of GC for subsequent transcriptomic analysis, the degradation of mRNA was diminished by using frozen tissues and by establishing a rapid staining protocol. This procedure enables histological and transcriptomic analysis of individual GC even within one lymphoid organ.

Original languageEnglish
Article numbere4431
JournalBio-protocol
Volume12
Issue number11
ISSN2331-8325
DOIs
Publication statusPublished - 05.06.2022

Research Areas and Centers

  • Academic Focus: Center for Infection and Inflammation Research (ZIEL)

DFG Research Classification Scheme

  • 205-33 Anatomy
  • 204-05 Immunology

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