title: Imaging effector memory T cells in the ear after induction of adoptive DTH.
abstract: Delayed type hypersensitivity (DTH) is an immune reaction in which the main players are CCR7(-) effector / memory T lymphocytes. Here, we demonstrate a method for inducing and recording the progress of a DTH reaction in the rat ear. This is followed by a demonstration of the preparation of rat ear tissue for two-photon imaging of the CCR7(-) effector / memory T cell response. An adoptive DTH is induced by the intraperitoneal injection of GFP-labeled Ova-specific CCR7(-) effector / memory T cell line (Beeton, C J. Visualized Experiments, Issue 8). Cells are then allowed to equilibrate in the rat for 48 hours before challenge by injecting one ear with saline (control ear) and the other with a 1:1 mix of Ova and Ova conjugated to Texas-Red (Ova-TR) to allow visualization of resident antigen-presenting cells. We describe a method of tissue preparation useful for imaging the motility of cells within the deep dermal layer during an immune response, in conjunction with visualization of collagen fibers by second harmonic generation. Ear tissue is cut into 5 x 5 mm squares (slightly larger is better) and mounted onto plastic cover slips using Vetbondtrade mark, which are then secured using silicone grease in an imaging chamber and superfused by oxygen-bubbled tissue culture medium at 37 degrees C.
authors.affiliation: Department of Physiology and Biophysics, University of California, Irvine, USA. mpmatheu@gmail.com
authors.forename: Melanie P
authors.initials: MP
authors.lastname: Matheu
authors.forename: Christine
authors.initials: C
authors.lastname: Beeton
authors.forename: Ian
authors.initials: I
authors.lastname: Parker
authors.forename: K George
authors.initials: KG
authors.lastname: Chandy
authors.forename: Michael
authors.initials: M
authors.lastname: D Cahalan
mesh_terms: D000818:Animals; D004431:Ear, External / Q000166:cytology / Q000276:immunology*; D005456:Fluorescent Dyes / Q000737:chemistry; D006968:Hypersensitivity, Delayed / Q000276:immunology*; D018796:Immunoconjugates / Q000737:chemistry / Q000276:immunology; D007156:Immunologic Memory; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D010047:Ovalbumin / Q000276:immunology; D051381:Rats; D054400:Receptors, CCR7 / Q000276:immunology; D012867:Skin / Q000166:cytology / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology*; D014966:Xanthenes / Q000737:chemistry
chemical_list: D005456:Fluorescent Dyes; D018796:Immunoconjugates; D054400:Receptors, CCR7; D014966:Xanthenes; C034657:Texas red; D010047:Ovalbumin