Technical advance: New in vitro method for assaying the migration of primary b cells using an endothelial monolayer as substrate

Phillip J. Stewart-Hutchinson, Taylor P. Szasz, Emily R. Jaeger, Michael D. Onken, John A. Cooper, Sharon Celeste Morley

Research output: Contribution to journalArticle

Abstract

Migration of B cells supports their development and recruitment into functional niches. Therefore, defining factors that control B cell migration will lead to a better understanding of adaptive immunity. In vitro cell migration assays with B cells have been limited by poor adhesion of cells to glass coated with adhesion molecules. We have developed a technique using monolayers of endothelial cells as the substrate for B cell migration and used this technique to establish a robust in vitro assay for B cell migration. We use TNF-α to up-regulate surface expression of the adhesion molecule VCAM-1 on endothelial cells. The ligand VLA-4 is expressed on B cells, allowing them to interact with the endothelial monolayer and migrate on its surface. We tested our new method by examining the role of L-plastin (LPL), an F-actin-bundling protein, in B cell migration. LPL-deficient (LPL-/-) B cells displayed decreased speed and increased arrest coefficient compared with wild-type (WT) B cells, following chemokine stimulation. However, the confinement ratios for WT and LPL-/- B cells were similar. Thus, we demonstrate how the use of endothelial monolayers as a substrate will support future interrogation of molecular pathways essential to B cell migration.

Original languageEnglish
Pages (from-to)941-948
Number of pages8
JournalJournal of Leukocyte Biology
Volume102
Issue number3
DOIs
StatePublished - Sep 2017

Keywords

  • Actin cytoskeleton
  • B cell migration
  • B lymphocytes
  • L-plastin

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