Imaging Effector Memory T cells in the Ear After Induction of Adoptive DTH

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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.

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Matheu, M. P., Beeton, C., Parker, I., Chandy, K. G., D. Cahalan, M. Imaging Effector Memory T cells in the Ear After Induction of Adoptive DTH. J. Vis. Exp. (18), e907, doi:10.3791/907 (2008).


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

  1. Euthanize the rat at the desired time-point by procedures outlined in your approved animal protocol. Here, we use an overdose of the anesthetic, isoflurane. This is done in a closed container located inside a well-ventilated hood. Ensure the animal is dead by either decapitation of by opening the thoracic cavity and cutting the heart.

  2. Using large scissors remove the ear with a single cut, as close to the body of the animal as possible (Figure 1).

    Figure 1. Removal of the rat ear

  3. Place the ear in a 15 ml conical tube containing ~10 ml of ice-cold 1x PBS or RPMI-1640. Keep the tissue samples on ice until you are ready to prepare the tissue for imaging.

  4. Image the tissue as soon as possible; we find, though, that 1 to 2 hours on ice has no discernable effect on cell motility in the tissue when compared to tissue harvested and imaged immediately.

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.

  1. Place both the tissue and media from the conical tube in a 10 cm tissue culture dish.

  2. With gloved hands hold the rat ear gently at the tip of the ear (distal) with the dorsal side of the ear facing up (Figure 2).

    Figure 2. Trim fur off the ear

  3. With your thumb on top of the ear, steady the tissue while using the tip of closed Dumont #5 scissors to separate the dermis from the deep dermis. Alternatively, if the edge of the ear has an overhang of skin/deep dermis, this can be grasped with forceps and gently pulled to separate this from the tissue underneath (Figure 3).

    Figure 3. Removal of the upper dermis

  4. Once a small area of skin has been separated from the underlying tissue, repeat the separation steps alternatively using scissors to cut between the dermal layers and forceps to gently remove and peel off the dislodged skin.

  5. Expose a 5 x 5 mm or larger area of ear tissue that is at least 3 mm from the initial site of antigen injection (Figure 4).

    Figure 4. Tissue exposed for imaging

II. Securing Tissue to the Imaging Stage

  1. Cut a plastic cover slip to a size slightly larger than prepared tissue.

  2. Spread a thin layer of 3M Vetbond™ tissue-safe glue across the slide.

  3. Grasp the corner of the prepared tissue (where you are not likely to image), remove from media, and dab the underside of the tissue on lens paper or Kym wipe to remove excess media.

  4. Touch the edge of the tissue to the glue covered slide (the Vetbond cures immediately on wet tissue). Gently stretch the tissue flat, with the opposite end being the last to touch the slide (Figure 5)

    Figure 5. Mount tissue to cover slip

  5. Cure the Vetbond™ by inverting the tissue sample/slide and dipping it in a reservoir of media such that the tissue and slide are face down and roughly at a 45 degree angle. Holding the tissue upside down at an angle helps to prevent excess glue from curing on the exposed tissue surface. Glue on the surface of the tissue will block the laser (Figure 6).

    Figure 6. Cure vetbond tissue glue by dipping in media

    Note: Steps 2-5 may take a little practice to master. Try practicing with discarded or extra tissue pieces before attempting for the first time.

  6. Dab a small amount of silicone grease onto the bottom of the slide.

  7. Place the slide in the perfusion chamber. Make sure that the perfusion media is flowing, 37°C and oxygenated before adding tissue. Gently press down on the edges of the slide making a seal with the silicon grease between the bottom of the perfusion chamber and the plastic cover slip.

III. Two-Photon Imaging

  1. Using bright-field transmitted light, focus on the top of the tissue. Turn off all of lights.

  2. Turn on the laser, PMTs as required by your multi-photon microscope system.

  3. Second harmonic generation (SHG) in highly-ordered fibrous proteins (collagen and myosin) produces a signal at half the wavelength of the excitation laser. We use excitation at 900 nM, leading to SHG at 450 nm which can be visualized using the "blue" channel of the microscope. SHG generation is a non-linear (two-photon) process, and thus provides an optical "sectioning" effect similar to that of two-photon excitation of fluorescence. The peak efficiency of SHG occurs when the fibrils are perpendicular to the laser, while fibrils that run in parallel will not be visualized.1

  4. Locate a good imaging area with plenty of cells, set your acquisition area such that the majority of cells are in the center and start imaging. Note: In DTH reactions, T cells and other cell infiltrates are often found to be concentrated together while other areas are devoid of infiltrating cells2. Therefore, it may take some time searching the tissue to find the best area to image.

  5. Check for cell viability by assessing cell velocity and polarity during imaging. Use the lowest laser power that provides acceptable image quality to ensure minimal photo-damage.

  6. Swap in a new tissue preparation as needed.

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  1. Koo, G. C., et al. Blockade of the voltage-gated potassium channel Kv1.3 inhibits immune responses in vivo. J Immunol. 158, 5120-5128 (1997).
  2. Gaga, M., Frew, A. J., Varney, V. A., Kay, A. B. Eosinophil activation and T lymphocyte infiltration in allergen-induced late phase skin reactions and classical delayed-type hypersensitivity. J Immunol. 147, 816-822 (1991).
  3. Flugel, A., Odoardi, F., Nosov, M., Kawakami, N. Autoaggressive effector T cells in the course of experimental autoimmune encephalomyelitis visualized in the light of two-photon microscopy. J Neuroimmunol. 191, 86-97 (2007).
  4. Kawakami, N., et al. Live imaging of effector cell trafficking and autoantigen recognition within the unfolding autoimmune encephalomyelitis lesion. J Exp Med. 201, 1805-1814 (2005).
  5. Miller, M. J., Wei, S. H., Parker, I., Cahalan, M. D. Two-photon imaging of lymphocyte motility and antigen response in intact lymph node. Science. 296, 1869-1873 (2002).
  6. Matheu, M. P. B., A, C. G. arcia, Chi, V., Rangaraju, S., Safrina, O., Monaghan, K., Uemura, M. I., Li, D., Pal, S., Monuki, E., Flugel, A., Pennington, M. W., Parker, I., Chandy, G. K., Calahan, M. D. In Situ Imaging of Effector/Memory T Cells During DTH and Suppression by Kv1.3 Channel Block. Immunity. In Press (2008).



  1. Hello. That was a great explanation for adoptive transfer and the techniques associated with that. In our laboratory, we are trying to employ DTH mouse model but we are using footpad, instead of an ear as it was shown that ear is less sensitive to linked suppression that footpad (W. J. Burlingham et. al.) We just acquired the same micrometer (thickness gauge) but our reading are very inconsistent. I was wondering if you ever tried footpad and could share some methods/hints to get stable/reproducible measurements.  Thanks, Vyacheslav Palchevskiy, Ph.D.

    Posted by: Anonymous
    November 3, 2008 - 10:27 PM
  2. Dear Vyacheslav, we have done a few trials in the footpad and have also had difficulties getting consistent results. I think it is because this area contains more soft tissue than the ear. One thing that may help: make sure your injections are very consistent in terms of amount and location (including depth). We have found that the depth of injection can considerably change the amount of inflammation and the number of T cells in the tissue. This is not a problem in the ear as you can’t really go too deep there. We always take at least 6 serial measurements for each ear or paw and then calculate a mean to try and get rid of the variation. The more variation we have, the more measurements we take of each ear or paw. Hope this help! Christine

    Posted by: Christine B.
    November 11, 2008 - 4:52 PM
  3. We are in agreement  that the mouse footpad presents technical problems, even for routine histology. We use footpad for adoptive transfer of human PBMC because this site seems more sensitive for detecting regulation; however if Vyacheslav only wants to study effector response the mouse ear works fine. Your imaging system looks very appealing . We will try in ear first, then later in footpad. Thanks for this very nice 'webinar'. Cheers--Will Burlingham, UW-Madison, WI

    Posted by: Anonymous
    December 5, 2008 - 3:41 PM
  4. Christine, I see you are using an Olympus ²0x dipping lens--is your ²photon system breadboard or a commercial Olympus? Right after you focus the objective into the ear tissue, the video switchs to the fluorescence time lapse.  Do you cover your sample/microscope or is your room very very dark.  Are you using non-descanned detectors?  Reason I am asking is on my ²004 BioRad MP system I have to cover the sample, monitors etc in order to use the nondesanned detectors.  It is hard for me to try these studies as I am also on an inverted system.  What wavelength are you using? Lance Rodenkirch UW

    Posted by: Anonymous
    December 10, 2008 - 5:13 PM
  5. Hi Lance, This is Melanie. We use a custom breadboard for our two photon scan head system, the detailed plans for our custom system are on the Parker Laboratory (UC Irvine) website. We both keep the room dark and cover our microscope and PMT detectors with black-out curtain to reduce noise from stray light. In order to manipulate the sample after this we work "blind" with our hands under the black-out curtain. Our PMT detectors are also mounted inside sealed aluminum box. To reduce stray light from the monitors from affecting the sample we have them at an angle facing slightly away from where the microscope and PMTs are, but we do not cover them. To visualize collagen and GFP at the same time we use between 890 and 9²0nM. 9²0nM is typically the wavelength we used. I hope this helps. Do check the Parker Lab website for more details on the system. Best of luck with your experiments! Melanie  

    Posted by: Melanie M.
    January 20, 2009 - 11:29 AM
  6. Great demo. We can employ this to our Imm. Lab. for the medical students. Can we acquire reagents from you girls and try it out here in Mackay Medical College, Taiwan.

    Posted by: Nan Chi C.
    April 20, 2011 - 5:46 PM

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