Here we demonstrate a method for inducing and recording the progress of a delayed type-hypersensitivity (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 effector / memory T cell response.
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 Vetbond™, which are then secured using silicone grease in an imaging chamber and superfused by oxygen-bubbled tissue culture medium at 37°C.
Induction of Adoptive DTH
Please see JoVE article: Induction and Monitoring of Adoptive Delayed-Type Hypersensitivity in Rats
Christine Beeton, George K. Chandy
Department of Physiology and Biophysics, University of California, Irvine
J. Visualized Experiments, Issue 8
Here, we have modified this protocol by using antigen (Ovalbumin) conjugated to Texas-Red in a 1:1 ratio with unlabeled antigen to allow for visualization of phagocytic antigen-presenting cells.
Ear Tissue Harvest
Tissue Preparation for Imaging
I. Removal of Epidermal and Dermal Layer
*Note: The deep dermal layer must be kept intact. If done correctly large blood vessels will remain intact and the cartilage of the ear will not be exposed. This is a difficult technique, especially when there is no inflammation (control conditions) and it is helpful to use a dissecting microscope.
II. Securing Tissue to the Imaging Stage
III. Two-Photon Imaging