Name: intravital imaging of intraepithelial lymphocytes in murine small intestine
Uploaded: 2019-06-24T15:00:00
Description: Watch this Scientific Journal Video about Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine at JoVE.com

This work is supported by NIH R21 AI143892, New Jersey Health Foundation Grant, Busch Biomedical Grant (KLE). We thank Madeleine Hu for her assistance in editing the manuscript and providing the data shown in the representative results.

All studies were conducted in an Association of the Assessment and Accreditation of Laboratory Animal Care (AALAC)-accredited facility according to protocols approved by Rutgers New Jersey Medical School Comparative Medicine Resources.
1. Mouse Preparation
NOTE: The following procedure, including animal preparation and surgery, will take 30&#8211;40 min. Prior to the surgery, turn on the microscope and warm up the enclosed incubator on the microscope ...

<ol>
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The development of intravital microscopy techniques has provided an unprecedented opportunity to observe the reorganization of subcellular structures8,9,22, cell-cell interactions12,25 and cell migratory behavior13,14,15,16,<sup clas...

Intraepithelial lymphocytes (IEL) are located within the intestinal epithelium, and are found both along the basement membrane and between adjacent epithelial cells in the lateral intercellular space1. There is approximately one IEL for every 5-10 epithelial cells; these IELs serve as sentinels to provide immune surveillance of the large expanse of the intestinal epithelial barrier2. IELs expressing the &#947;&#948; T cell receptor (TCR) comprise up to 60% of the total IEL population in the murine small intestine. Studies in &#947;&#948; T-cell-deficient mice demonstrate a largely protective role of these cells in response to intestinal injury, inflammation and infection3,4,5. Despite the generation of the Tcrd knockout mouse6, our understanding of &#947;&#948; IEL biology remains limited due in part to the fact that ligands recognized by the &#947;&#948; TCR have yet to be identified7. As a result, the lack of tools to study this cell population has made it difficult to investigate the role of &#947;&#948; TCR activation and function under physiological and pathological conditions. To fill this gap, we have developed live imaging techniques to visualize &#947;&#948; IEL migratory behavior and interactions with neighboring enterocytes as a means to provide additional insight into &#947;&#948; IEL function and responsiveness to external stimuli in vivo.
Over the last decade, intravital imaging has significantly expanded our understanding of the molecular events involved in multiple facets of intestinal biology, including epithelial cell shedding8, regulation of epithelial barrier function9,10, myeloid cell sampling of luminal contents11,12, and host-microbe interactions11,13,14,15,16. In the context of IEL biology, the use of intravital microscopy has shed light on the spatiotemporal dynamics of IEL motility and the factors mediating their surveillance behavior13,14,15,16. The development of TcrdH2BeGFP (TcrdEGFP) reporter mice, which labels &#947;&#948; IELs by nuclear GFP expression17, revealed that &#947;&#948; IELs are highly motile within the epithelium and exhibit a unique surveillance behavior that is responsive to microbial infection17,13,14. Recently, another &#947;&#948; T cell reporter mouse was developed (Tcrd-GDL) which expresses GFP in the cytoplasm to allow visualization of the entire cell18. Similar methodology has been used to investigate the requirement of specific chemokine receptors, such as G protein-coupled receptor (GPCR)-18 and -55, on the dynamics of IEL motility19,20. In the absence of a cell-specific reporter, fluorescent conjugated antibodies against CD8&#945; were used to visualize and track IEL motility in vivo19,20. Although two-photon laser scanning microscopy is commonly used for intravital imaging, the use of spinning disk confocal laser microscopy provides unique advantages to capture high speed and high-resolution multi-channel images with minimal background noise. This technology is ideal to elucidate the spatiotemporal dynamics of immune/epithelial interactions within the complex microenvironment of the intestinal mucosa. Moreover, through the use of various transgenic and/or knockout mouse models, these studies can provide insight into the molecular regulation of intestinal immune and/or epithelial cell function.

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			<td>MatTek</td>
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			<td>Alexa Fluor 633 Hydrazide</td>
			<td>Invitrogen</td>
			<td>A30634</td>
			<td></td>
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			<td>BD PrecisionGlide Hypodermic needles - 27g</td>
			<td>Thermo Fisher Scientific</td>
			<td>14-826-48</td>
			<td></td>
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			<td>BD Slip Tip Sterile Syringe - 1 ml</td>
			<td>Thermo Fisher Scientific</td>
			<td>14-823-434</td>
			<td></td>
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		<tr>
			<td>BD Tuberculin Syringe</td>
			<td>Thermo Fisher Scientific</td>
			<td>14-829-9</td>
			<td></td>
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			<td>Dissecting scissors</td>
			<td>Thermo Fisher Scientific</td>
			<td>08-940</td>
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			<td>Electrocautery</td>
			<td>Thermo Fisher Scientific</td>
			<td>50822501</td>
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			<td>Enclosed incubation chamber</td>
			<td>OKOLAB</td>
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			<td>Microscope</td>
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			<td>Eye Needles, Size #3; 1/2 Circle, Taper Point, 12 mm Chord Length</td>
			<td>Roboz</td>
			<td>RS-7983-3</td>
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			<td>Hank&#39;s Balanced Salt Solution</td>
			<td>Sigma-Aldrich</td>
			<td>55037C</td>
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			<td>Hoechst 33342</td>
			<td>Invitrogen</td>
			<td>H3570</td>
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			<td>Imaris (v. 9.2.1) with Start, Track, XT modules</td>
			<td>Bitplane</td>
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			<td>Software</td>
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			<td>Inverted DMi8</td>
			<td>Leica</td>
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			<td>IQ3 (v. 3.6.3)</td>
			<td>Andor</td>
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			<td>VWR</td>
			<td>21905-026</td>
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			<td>McPherson-Vannas scissors 3&#8221; (7.5 cm) Long 5X0.15mm Straight Sharp</td>
			<td>Roboz</td>
			<td>RS-5600</td>
			<td></td>
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			<td>Non-absorbable surgical suture, Silk Spool, Black Braided</td>
			<td>Fisher Scientific</td>
			<td>NC0798934</td>
			<td></td>
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			<td>Nugent Forceps 4.25&#8221; (11 cm) Long Angled Smooth 1.2mm Tip</td>
			<td>Roboz</td>
			<td>RS-5228</td>
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			<td>Puralube Vet Ointment</td>
			<td>Dechra</td>
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			<td>Lubricating Eye Ointment</td>
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			<td>Spinning disk Yokogawa CSU-W1 with a 63x 1.3 N.A. HC PLAN APO glycerol immersion objective, iXon Life 888 EMCCD camera, 405 nm diode laser, 488 nm DPSS laser, 640 nm diode laser</td>
			<td>Andor</td>
			<td></td>
			<td>Confocal system</td>
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			<td>Xylazine</td>
			<td>Akorn</td>
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			<td>Anesthesia</td>
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intravital imaging of intraepithelial lymphocytes in murine small intestine

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal epithelium. Due in part to the lack of a definitive ligand for the &#947;&#948; T cell receptor, our understanding of the regulation of &#947;&#948; IEL activation and their function in vivo remains limited. This necessitates the development of alternative strategies to interrogate signaling pathways involved in regulating &#947;&#948; IEL function and the responsiveness of these cells to the local microenvironment. Although &#947;&#948; IELs are widely understood to limit pathogen translocation, the use of intravital imaging has been critical to understanding the spatiotemporal dynamics of IEL/epithelial interactions at steady-state and in response to invasive pathogens. Herein, we present a protocol for visualizing IEL migratory behavior in the small intestinal mucosa of a GFP &#947;&#948; T cell reporter mouse using inverted spinning disk confocal laser microscopy. Although the maximum imaging depth of this approach is limited relative to the use of two-photon laser-scanning microscopy, spinning disk confocal laser microscopy provides the advantage of high speed image acquisition with reduced photobleaching and photodamage. Using 4D image analysis software, T cell surveillance behavior and their interactions with neighboring cells can be analyzed following experimental manipulation to provide additional insight into IEL activation and function within the intestinal mucosa.

Using intravital imaging of TcrdEGFP reporter mice, we have previously shown that &#947;&#948; IELs exhibit a dynamic surveillance behavior, in which they patrol the epithelium by migrating along the basement membrane and into the lateral intercellular space (LIS) at steady state (Figure 2, Movie 1).
This approach can also be used to evaluate how the inhibition of specific cell signaling pathways and/or receptors influences &#947;&#948; IEL migrat...

Watch this Scientific Journal Video about Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine at JoVE.com

Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

We describe a method to visualize GFP-labeled &#947;&#948; IELs using intravital imaging of murine small intestine by inverted spinning disk confocal microscopy. This technique enables the tracking of live cells within the mucosa for up to 4 h and can be used to investigate a variety of intestinal immune-epithelial interactions. &#160;

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal ...

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