Visualization of integrin Mac-1 in vivo

J Immunol Methods. 2015 Nov;426:120-7. doi: 10.1016/j.jim.2015.08.012. Epub 2015 Sep 2.

Abstract

β2 integrins play critical roles in migration of immune cells and in the interaction with other cells, pathogens, and the extracellular matrix. Among the β2 integrins, Mac-1 (Macrophage antigen-1), composed of CD11b and CD18, is mainly expressed in innate immune cells and plays a major role in cell migration and trafficking. In order to image Mac-1-expressing cells both in live cells and mouse, we generated a knock-in (KI) mouse strain expressing CD11b conjugated with monomeric yellow fluorescent protein (mYFP). Expression of CD11b-mYFP protein was confirmed by Western blot and silver staining of CD11b-immunoprecipitates and total cell lysates from the mouse splenocytes. Mac-1-mediated functions of the KI neutrophils were comparable with those in WT cells. The fluorescence intensity of CD11b-mYFP was sufficient to image CD11b expressing cells in live mice using intravital two-photon microscopy. In vitro, dynamic changes in the intracellular localization of CD11b molecules could be measured by epifluorescent microscopy. Finally, CD11b-expressing immune cells from tissue were easily detected by flow cytometry without anti-CD11b antibody staining.

Keywords: CD11b; Imaging; Integrin; MAC-1; YFP.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Bacterial Proteins / genetics
  • CD11b Antigen / immunology*
  • CD18 Antigens / immunology
  • Cell Adhesion
  • Cell Movement / immunology
  • Extracellular Matrix / immunology
  • Flow Cytometry
  • Gene Knock-In Techniques
  • Intercellular Adhesion Molecule-1 / metabolism
  • Luminescent Proteins / genetics
  • Macrophage-1 Antigen / analysis*
  • Macrophage-1 Antigen / immunology*
  • Mice
  • Mice, Inbred C57BL
  • Mice, Transgenic
  • Microscopy, Fluorescence
  • Neutrophils / immunology*
  • Phagocytosis / immunology
  • Spleen / cytology*
  • Spleen / immunology
  • Staining and Labeling / methods

Substances

  • Bacterial Proteins
  • CD11b Antigen
  • CD18 Antigens
  • Luminescent Proteins
  • Macrophage-1 Antigen
  • yellow fluorescent protein, Bacteria
  • Intercellular Adhesion Molecule-1