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An investigation into the profile and dynamics of neutrophil transendothelial cell migration (TEM) using high resolution in vivo real‐time confocal imaging
Author(s) -
Woodfin Abigail,
Voisin MathieuBenoit,
Colom Bartomeu,
Nourshargh Sussan
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.232.2
Subject(s) - transcellular , paracellular transport , in vivo , confocal , confocal microscopy , biophysics , microbiology and biotechnology , chemistry , intravital microscopy , live cell imaging , cell , biology , permeability (electromagnetism) , biochemistry , physics , membrane , optics
A high resolution real‐time 3D imaging technique was established for analyzing the characteristics and dynamics of neutrophil transmigration in vivo . Our model involves rapid confocal microscopy imaging of the cremaster muscle of lys‐ EGFP mice (which exhibit EGFP + ‐neutrophils) in conjunction with fluorescently labeled endothelial cell (EC) junctions using an anti‐PECAM‐1 mAb. With this method we have examined the profile and dynamics of neutrophil TEM as elicited by several stimuli (eg IL‐1β, FMLP and ischemia/reperfusion injury). In all reactions the majority of the TEM events occurred via the paracellular route where the formation of EC junctional pores at bi‐ and tri‐cellular junctions could be clearly imaged and analyzed in terms of their frequency (no significant difference noted between bi‐ and tri‐cellular junctions) and duration (6 ± 0.5 minutes in IL‐1β‐stimulated tissues). Clear evidence for the formation of transcellular pores was also obtained (9.3 ± 2.1 % for all reactions). Collectively the high spatial and temporal resolution of our approach has led to novel qualitative and quantitative analysis of neutrophil TEM in vivo . Funded by the Wellcome Trust (Ref: 081172/Z/06/Z to SN)