Open AccessProtocol to Isolate Germinal Centers by Laser Microdissection
Author(s) -
Farbod Bahreini,
Markus Niebuhr,
Julia Belde,
Jürgen Westermann,
Kathrin Kalies
Publication year - 2022
Publication title -
bio-protocol
Language(s) - Uncategorized
Resource type - Journals
ISSN - 2331-8325
DOI - 10.21769/bioprotoc.4431
Subject(s) - germinal center , laser capture microdissection , flow cytometry , biology , microbiology and biotechnology , microdissection , antigen , transcriptome , follicular dendritic cells , chemistry , antigen presenting cell , immune system , b cell , t cell , immunology , gene expression , antibody , gene , genetics
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.