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 to 37 &#176;C.
<ol>
	<li>Perform experiments on 8&#8211;10 weeks old TcrdEGFP mice on a C57BL/6 background, which were obtained from Bernard Malissen (INSERM, Paris, France). According to IACUC-approved procedure, anesthetize the mouse by i.p. injection of ketamine (120 mg/kg) and xylazine (10 mg/kg) based on the weight of the animal. Evaluate the level of anesthesia by respiration rate (55&#8211;65 breaths per minute)21, toe pinch and palpebral reflex.</li>
	<li>Once surgical plane anesthesia is reached, secure the hind legs of the mouse using tape to a heating pad to maintain body temperature, which can be monitored by rectal thermometer. If there are signs that the anesthesia is wearing off (e.g., increased respiration rate, blinking and/or eye muscle twitching response to palpebral reflex), re-administer 50&#160;&#956;L of ketamine/xylazine at a time until the mouse is fully sedated.</li>
	<li>Prepare the nuclear dye by diluting 50 &#181;L of Hoechst 33342 (10 mg/mL) with 315 &#181;L of 0.9% saline; the final concentration is 37.5 mg/kg based on the weight of the mouse (200 &#181;L approximate volume). Once the mouse is anesthetized, slowly inject the Hoechst using a tuberculin syringe through the retro-orbital venous sinus. 
	NOTE: Wait 1&#8211;2 min before proceeding to the next step to allow the mouse to acclimate to the change in circulating volume following retro-orbital injection.</li>
	<li>Place a small amount of lubricant over the eyes to prevent them from drying out.</li>
</ol>
2. Mouse Surgery: Laparotomy to Expose Intestinal Mucosa
<ol>
	<li>Make a 2 cm vertical incision through skin and peritoneum along the midline of the lower abdomen to externalize the region of intestine to be imaged.</li>
	<li>Using angled forceps, carefully pull the cecum out of the peritoneal cavity and identify the area of small intestine to be imaged. Be careful not to tear mesenteric blood vessels during this process. To minimize potential damage or bleeding, avoid grabbing or pinching the intestine with the forceps.</li>
	<li>Locate a suitable region of small intestine (2&#8211;3 cm) for imaging that contains minimal fecal contents. Carefully return the cecum and remaining small intestine into the peritoneal cavity, while leaving the segment of interest externalized. 
	NOTE: Avoid puncturing the cecum or disrupting the mesenteric vasculature during this step.</li>
	<li>Place two pairs of forceps on either side of the underlying mesentery between blood vessels, and gently rub the tips of the forceps together to create a hole in the membrane (Figure 1)22. This will allow the needle to pass through the mesentery when closing the peritoneal cavity beneath the intestine.</li>
	<li>Close the incision while keeping the loop of intestine externalized. Using angled forceps and a curved, taper point needle attached to a 5 cm suture, penetrate one side of the peritoneum, go through the hole made in the previous step, and up through the other side of the peritoneal lining. Place one suture at the top, and another near the bottom of the incision. 
	NOTE: Avoid damaging the vasculature during this step.</li>
	<li>Repeat this process to close the skin beneath the intestinal loop, by placing one suture in the middle of the incision, between the previous sutures in the underlying peritoneum. 
	NOTE: It is important to only externalize the area that is to be imaged; this will reduce extraneous motion during imaging. Be sure to avoid tying off the mesenteric arteries.</li>
	<li>Use an electrocautery to make a line of perforation along the anti-mesenteric border. Immediately after cauterization, apply a few drops of water to the surface of the intestine to prevent additional heat-induced tissue damage. Blot with a Kimwipe to remove residual water.</li>
	<li>Cut a small horizontal slit at the distal edge of the cauterized tissue using Vannas scissors and proceed to cut along the length of the cauterized line toward the proximal end of the externalized tissue segment (approximately 1.5 cm in length) to expose the mucosal surface. 
	NOTE: If necessary, use forceps to gently remove any excess fecal content remaining on the exposed mucosal surface.</li>
	<li>Cover the abdomen of the mouse with a moist Kimwipe to prevent the tissue from becoming dehydrated. Transport the mouse to the microscope in a covered vessel.</li>
</ol>
3. Image Acquisition by Spinning Disk Confocal Microscopy
<ol>
	<li>Pipette 150 &#181;L of 1 &#181;M AlexaFluor 633 in HBSS onto the glass coverslipped bottom of a 35 mm dish. Position the mouse so that the opened mucosal surface directly contacts the coverslip.</li>
	<li>Tilt the head of the microscope back and place the mouse on the coverslipped dish on the imaging stage in a pre-warmed incubator. Alternatively, cover the mouse by a heating blanket to maintain body temperature.</li>
	<li>Launch imaging software. The excitation intensity and exposure time for each laser should not exceed 10&#8211;15 mW or 120&#8211;150 ms, respectively, to avoid photobleaching and to minimize the interval between time points. Adjust the frame average to &#8220;2&#8221; and turn on the EM gain function to reduce background noise. Select 63x objective calibration to ensure the correct measurement of pixel size. 
	NOTE: The settings detailed above are meant to provide an initial reference, but will vary depending on individual microscope configurations.</li>
	<li>Using the 405 nm laser and the 20x air objective, visualize the nuclei to locate a field of villi that lack noticeable movement or drift by eye. Avoid areas of artifactual movement due to respiration, peristalsis or heartbeat.</li>
	<li>Using the XY scan, record the XY coordinate of the field of interest and switch to the glycerol immersion 63X objective.</li>
	<li>Confirm that the villi in the selected field are stable by acquiring a live image on the 405 nm channel for up to 1 min.</li>
	<li>While acquiring a live image on the 405 nm channel, adjust the focus to find the orthogonal plane just beneath the villous tip. Acquire Z-stacks starting from the just below the villous tip epithelium down the villus until it is difficult to resolve the nuclei, about 15&#8211;20 &#181;m, using a 1.5 &#181;m step. 
	NOTE: When imaging with three channels (405, 488, 640 nm) using the exposure times described above, it is possible to acquire all three channels sequentially at approximately 20 s intervals.</li>
	<li>Open the software in analysis mode, 3&#8211;5 min after beginning the acquisition, to confirm the image stability and &#947;&#948; IEL motility. Continue to acquire images for 30&#8211;60 min for each field of villi. Record 2&#8211;3 fields for each mouse. While imaging, monitor the mouse every 5 min. 
	NOTE: If the anesthesia begins to wear off, use forceps to gently scruff the neck of the mouse to administer 50 &#181;L of ketamine/xylazine at a time. Continue to monitor anesthesia levels and re-administer when necessary.</li>
	<li>It is not advisable to image a single mouse for longer than 4 h. Once image acquisition is complete, sacrifice the mice by cervical dislocation followed by bilateral pneumothorax.</li>
</ol>
4. 4D Analysis of Images
<ol>
	<li>Directly import imaging files to 4D rendering software.</li>
	<li>Create individual Surfaces for IELs and the lumen, then track the surfaces over time using the parameters outlined in Table 1 as an initial reference. For the IELs, enable Split touching objects using the seed point diameter indicated.</li>
	<li>Use the autoregressive tracking algorithm to determine IEL motility. Although the tracking algorithm is relatively accurate, it is imperative to visually inspect tracks for each individual IEL. If a track is incorrect, correct it using the Disconnect and Connect functions under the Edit Tracks tab.</li>
	<li>To determine the distance from each IEL to the lumen, select the lumen Surface and perform Distance Transformation function under the Tool menu. When prompted, select Outside Surface. Once calculated, the distance transformation will be displayed as the 4th channel.</li>
	<li>Filter the Intensity min of the 4th channel to select &#947;&#948; IELs that are within the lateral intercellular space (LIS). This value should be close to 15 &#181;m, based on the height of a columnar epithelial cell. The percent of total &#947;&#948; IELs within this range are reported as the frequency of &#947;&#948; IELs in the LIS.</li>
	<li>For further analysis, export statistical data including: IEL track speed, track displacement, track straightness and track length. Alternatively, analyze data using the Vantage module. Depending on the experiment, obtain additional metrics from the acquired data, including dwell time within the LIS or IEL/epithelial interactions.</li>
</ol>

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The authors have nothing to disclose.

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.

<table>
	<tbody>
		<tr>
			<td>35mm dish, No. 1.5 Coverslip</td>
			<td>MatTek</td>
			<td>P35G-1.5-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 633 Hydrazide</td>
			<td>Invitrogen</td>
			<td>A30634</td>
			<td></td>
		</tr>
		<tr>
			<td>BD PrecisionGlide Hypodermic needles - 27g</td>
			<td>Thermo Fisher Scientific</td>
			<td>14-826-48</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Slip Tip Sterile Syringe - 1 ml</td>
			<td>Thermo Fisher Scientific</td>
			<td>14-823-434</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Tuberculin Syringe</td>
			<td>Thermo Fisher Scientific</td>
			<td>14-829-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting scissors</td>
			<td>Thermo Fisher Scientific</td>
			<td>08-940</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrocautery</td>
			<td>Thermo Fisher Scientific</td>
			<td>50822501</td>
			<td></td>
		</tr>
		<tr>
			<td>Enclosed incubation chamber</td>
			<td>OKOLAB</td>
			<td></td>
			<td>Microscope</td>
		</tr>
		<tr>
			<td>Eye Needles, Size #3; 1/2 Circle, Taper Point, 12 mm Chord Length</td>
			<td>Roboz</td>
			<td>RS-7983-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution</td>
			<td>Sigma-Aldrich</td>
			<td>55037C</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Invitrogen</td>
			<td>H3570</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris (v. 9.2.1) with Start, Track, XT modules</td>
			<td>Bitplane</td>
			<td></td>
			<td>Software</td>
		</tr>
		<tr>
			<td>Inverted DMi8</td>
			<td>Leica</td>
			<td></td>
			<td>Microscope</td>
		</tr>
		<tr>
			<td>IQ3 (v. 3.6.3)</td>
			<td>Andor</td>
			<td></td>
			<td>Software</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Putney</td>
			<td></td>
			<td>Anesthesia</td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>VWR</td>
			<td>21905-026</td>
			<td></td>
		</tr>
		<tr>
			<td>McPherson-Vannas scissors 3&#8221; (7.5 cm) Long 5X0.15mm Straight Sharp</td>
			<td>Roboz</td>
			<td>RS-5600</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-absorbable surgical suture, Silk Spool, Black Braided</td>
			<td>Fisher Scientific</td>
			<td>NC0798934</td>
			<td></td>
		</tr>
		<tr>
			<td>Nugent Forceps 4.25&#8221; (11 cm) Long Angled Smooth 1.2mm Tip</td>
			<td>Roboz</td>
			<td>RS-5228</td>
			<td></td>
		</tr>
		<tr>
			<td>Puralube Vet Ointment</td>
			<td>Dechra</td>
			<td></td>
			<td>Lubricating Eye Ointment</td>
		</tr>
		<tr>
			<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>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Akorn</td>
			<td></td>
			<td>Anesthesia</td>
		</tr>
	</tbody>
</table>

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 ...

intravital-imaging-intraepithelial-lymphocytes-murine-small

Research

JoVE Journal

Immunology and Infection

7.8K Views.  Rutgers New Jersey Medical School. We describe a method to visualize GFP-labeled γδ 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.  

Video: Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Intravital Imaging of the Mouse Popliteal Lymph Node

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign antigens from peripheral tissues to prime the adaptive immune response. Juxtaposed between innate and adaptive immune responses, the LN is an ideal site to study immune cell interactions1,2. Lymphocytes (T cells, B cells and NK cells), dendritic cells (DCs), and macrophages comprise the bulk of bone marrow-derived cellular elements of the LN. These cells are strategically positioned in the LN to allow efficient surveillance of self antigens and potential foreign antigens3-5. The process by which lymphocytes successfully encounter cognate antigens is a subject of intense investigation in recent years, and involves an integration of molecular contacts including antigen receptors, adhesion molecules, chemokines, and stromal structures such as the fibro-reticular network2,6-12. 
Prior to the development of high-resolution real-time fluorescent in vivo imaging, investigators relied on static imaging, which only offers answers regarding morphology, position, and architecture. While these questions are fundamental in our understanding of immune cell behavior, the limitations intrinsic with this technique does not permit analysis to decipher lymphocyte trafficking and environmental clues that affect dynamic cell behavior. Recently, the development of intravital two-photon laser scanning microscopy (2P-LSM) has allowed investigators to view the dynamic movements and interactions of individual cells within live LNs in situ12-16. In particular, we and others have applied this technique to image cellular behavior and interactions within the popliteal LN, where its compact, dense nature offers the advantage of multiplex data acquisition over a large tissue area with diverse tissue sub-structures11,17-18. It is important to note that this technique offers added benefits over explanted tissue imaging techniques, which require disruption of blood, lymph flow, and ultimately the cellular dynamics of the system. Additionally, explanted tissues have a very limited window of time in which the tissue remains viable for imaging after explant. With proper hydration and monitoring of the animal's environmental conditions, the imaging time can be significantly extended with this intravital technique. Here, we present a detailed method of preparing mouse popliteal LN for the purpose of performing intravital imaging.

Recent advances in 2-photon microscopy have enabled real-time in situ imaging of live tissues in animal models, thereby enhancing our ability to investigate cellular behavior in both physiologic and pathologic conditions. Here, we outline the preparations required to perform intravital imaging of the mouse popliteal lymph node.

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign ...

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches

Peyer's patches (PPs) are integral components of the gut-associated lymphoid tissues (GALT) and play a central role in intestinal immunosurveillance and homeostasis. Particulate antigens and microbes in the intestinal lumen are continuously sampled by PP M cells in the follicle-associated epithelium (FAE) and transported to an underlying network of dendritic cells (DCs), macrophages, and lymphocytes. In this article, we describe protocols in which murine PPs are (i) dissociated into single cell suspensions and subjected to flow cytometry and (ii) prepared for cryosectioning and immunostaining. For flow cytometry, PPs are mechanically dissociated and then filtered through 70 &mu;m membranes to generate single cell suspensions free of epithelial cells and large debris. Starting with 20-25 PPs (from four mice), this quick and reproducible method yields a population of &gt;2.5 x 106 cells with &gt;90% cell viability. For cryosectioning, freshly isolated PPs are immersed in Optimal Cutting Temperature (OCT) medium, snap-frozen in liquid nitrogen, and then sectioned using a cryomicrotome. Tissue sections (5-12 &mu;m) are air-dried, fixed with acetone or methanol, and then subjected to immunolabeling.

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer&#039;s Patches

There is an increasing interest in understanding the immunological functions of specific subpopulations of cells in Peyer's patches (PPs), the primary inductive sites of gut-associated lymphoid tissues. Here we outline parallel protocols for preparing PP single cell preparations for flow cytometric analysis and PP cryosections for immunostaining.

Peyer's  patches (PPs) are integral components of the gut-associated lymphoid tissues  (GALT) and play a central role in intestinal immunosurveillance and ...

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 &#181;m tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells &#8211; on the level of a few protein molecules in germinal centers.

High-resolution intravital imaging with enhanced contrast up to 120 &#181;m depth in lymph nodes of adult mice is achieved by spatially modulating the excitation pattern of a multi-focal two-photon microscope. In 100 &#181;m depth we measured resolutions of 487 nm (lateral) and 551 nm (axial), thus circumventing scattering and diffraction limits.

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick ...

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both microbial and dietary. Given an increasing understanding that these luminal antigens help shape the immune response and that education of immune cells within the intestine is critical for a number of systemic diseases, there has been increased interest in characterizing the intestinal immune system. However, many published protocols are arduous and time-consuming. We present here a simplified protocol for the isolation of lymphocytes from the small-intestinal lamina propria, intraepithelial layer, and Peyer&#39;s patches that is rapid, reproducible, and does not require laborious Percoll gradients. Although the protocol focuses on the small intestine, it is also suitable for analysis of the colon. Moreover, we highlight some aspects that may need additional optimization depending on the specific scientific question. This approach results in the isolation of large numbers of viable lymphocytes that can subsequently be used for flow cytometric analysis or alternate means of characterization.

There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte cell culture and 64Cu-antibody receptor targeting of the cells. In vitro evaluation of the radiolabel and non-invasive in vivo cell tracking in an animal model of an airway delayed-type hypersensitivity reaction (DTHR) by PET/CT are described.
In detail, the conjugation of a mAb with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is shown. Following the production of radioactive 64Cu, radiolabeling of the DOTA-conjugated mAb is described. Next, the expansion of chicken ovalbumin (cOVA)-specific CD4+ interferon (IFN)-&#947;-producing T helper cells (cOVA-TH1) and the subsequent radiolabeling of the cOVA-TH1 cells are depicted. Various in vitro techniques are presented to evaluate the effects of 64Cu-radiolabeling on the cells, such as the determination of cell viability by trypan blue exclusion, the staining for apoptosis with Annexin V for flow cytometry, and the assessment of functionality by IFN-&#947; enzyme-linked immunosorbent assay (ELISA). Furthermore, the determination of the radioactive uptake into the cells and the labeling stability are described in detail. This protocol further describes how to perform cell tracking studies in an animal model for an airway DTHR and, therefore, the induction of cOVA-induced acute airway DHTR in BALB/c mice is included. Finally, a robust PET/CT workflow including image acquisition, reconstruction, and analysis is presented.
The 64Cu-antibody receptor targeting approach with subsequent receptor internalization provides high specificity and stability, reduced cellular toxicity, and low efflux rates compared to common PET-tracers for cell labeling, e.g.64Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (64Cu-PTSM). Finally, our approach enables non-invasive in vivo cell tracking by PET/CT with an optimal signal-to-background ratio for 48 h. This experimental approach can be transferred to different animal models and cell types with membrane-bound receptors that are internalized.

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

Following the preparation of a 64Cu-modified monoclonal antibody binding to a transgenic murine T cell receptor, T cells are radiolabeled in vivo, analyzed for viability, functionality, labeling stability and apoptosis, and adoptively transferred into mice with an airway delayed-type hypersensitivity reaction for non-invasive imaging by positron emission tomography/computed tomography (PET/CT).

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte ...

Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are extremely high, no direct treatment or organ-related mechanism has been examined in detail in real time. The liver is the key organ that manages toxins and infections in the human body. Herein, we aimed to perform intravital imaging of mouse liver after induction of endotoxemia in order to track the motility of immune cells, such as neutrophils and liver capsular macrophages (LCMs). Accordingly, we designed a novel surgical method for exposure of the liver with minimally invasive surgery. Mice were intraperitoneally injected with lipopolysaccharide (LPS), a common endotoxin. Using our novel surgical approach for exposure and intravital imaging of the mouse liver, we found that neutrophil recruitment in LPS-treated LysM-green fluorescent protein (GFP) mouse liver was increased compared with that in phosphate-buffered saline-treated liver. After LPS treatment, the number of neutrophils increased significantly with time. Additionally, using CX3Cr1-GFP mice, we successfully visualized liver resident macrophages called LCMs. Therefore, to investigate the efficacy of new reagents to control immune mobility in vivo, determining the motility and morphology of neutrophils and LCMs in the liver may allow us to identify therapeutic effect in organ failure and tissue damage caused by leukocytes activation in sepsis.

We established a novel surgical protocol for two-photon imaging of live mice liver with minimal invasion. With this technique, we identified the detailed structure of the liver during lipopolysaccharide-induced endotoxemia. We anticipate that this method may be utilized to determine the effectiveness of various reagents treatment to hepatic leukocyte migration.

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...