Visualization of integrin Mac-1 in vivo.
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.
Department of Microbiology and Immunology, David H. Smith Center for Vaccine Biology and Immunology, University of Rochester, Rochester, NY, USA. Electronic address: Kihong_Lim@urmc.rochester.edu.
Kihong
K
Lim
Department of Microbiology and Immunology, David H. Smith Center for Vaccine Biology and Immunology, University of Rochester, Rochester, NY, USA.
Young-Min
YM
Hyun
Department of Microbiology and Immunology, David H. Smith Center for Vaccine Biology and Immunology, University of Rochester, Rochester, NY, USA.
Kris
K
Lambert-Emo
Department of Microbiology and Immunology, David H. Smith Center for Vaccine Biology and Immunology, University of Rochester, Rochester, NY, USA.
David J
DJ
Topham
Department of Microbiology and Immunology, David H. Smith Center for Vaccine Biology and Immunology, University of Rochester, Rochester, NY, USA. Electronic address: minsoo_kim@urmc.rochester.edu.
Minsoo
M
Kim
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D039481:CD11b Antigen / Q000276:immunology*; D018821:CD18 Antigens / Q000276:immunology; D002448:Cell Adhesion; D002465:Cell Movement / Q000276:immunology; D005109:Extracellular Matrix / Q000276:immunology; D005434:Flow Cytometry; D055879:Gene Knock-In Techniques; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D016177:Macrophage-1 Antigen / Q000032:analysis* / Q000276:immunology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence; D009504:Neutrophils / Q000276:immunology*; D010587:Phagocytosis / Q000276:immunology; D013154:Spleen / Q000166:cytology* / Q000276:immunology; D013194:Staining and Labeling / Q000379:methods
D001426:Bacterial Proteins; D039481:CD11b Antigen; D018821:CD18 Antigens; D008164:Luminescent Proteins; D016177:Macrophage-1 Antigen; C054562:yellow fluorescent protein, Bacteria; D018799:Intercellular Adhesion Molecule-1
CD11b; Imaging; Integrin; MAC-1; YFP