Name: imaging cd4 t cell interstitial migration in the inflamed dermis
Uploaded: 2016-03-25T14:00:00.000+00:00
Duration: 11 min 28 s
Description: Watch this Scientific Journal Video about Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis at JoVE.com

作者感谢罗切斯特多光子显微镜核心设施的大学与实时成像帮助。支持NIH AI072690和AI02851到DJF; AI114036以股份公司和AI089079到MGO。

意义 这里，我们提出一个完整的协议为传输，抗原特异性效应Th1细胞在完整小鼠耳真皮4D可视化。此方法提供了以下几个原因一些当前成像模式的优点。通过成像腹耳真皮，我们能够放弃所需的涉及其他部位皮肤成像方案脱毛。虽然脱毛剂通常是温和的，它们已被证明导致破坏皮肤屏障42，这个过程可以刺激免疫应答43,44。还通过避免侵入外科手术以暴...

The effector function of CD4 T cells is critically dependent on their ability to rapidly enter and traverse a wide variety of peripheral tissues to survey for damage, locate foci of infection, or cause pathology from chronic infection or autoimmunity. While the processes of homing to inflamed sites1-4 and extravasation5-7 from the vasculature into tissues have been well-characterized, the factors that drive and regulate the interstitial motility of T cells remain undefined. The migration of T cells in complex 3D environments has been studied in vitro through the use of artificial matrices8-10 or microfluidic devices11,12, but these fail to recapitulate the complex and dynamic environment of an in vivo system. It is only recently, with the advent of high-resolution multi-color intravital imaging that it has become possible to study the dynamic behavior of immune cells in situ, allowing for a better understanding of intact immune responses.
Over a decade ago, several influential studies were published that first utilized multiphoton microscopy to address immunological questions. Early studies focused on the behavior of immune cells within explanted lymphoid organs13-16, which were soon followed by techniques to image exposed lymph nodes in anesthetized mice17. Imaging allowed for new fundamental observations about the stages of lymph node priming of T cells18, the mechanisms by which T cells migrate in secondary lymphoid organs19, T cell interactions with other immune cells20,21, and dynamic T cell positioning within the lymph node22. Although many early studies focused on lymph node dynamics, intravital imaging has been since been utilized to image the immune response in many peripheral tissues, including the brain23-25, liver26, lung27, and skin28-30. 
The mouse ear dermis is particularly well poised for imaging, due to the thinness of ear skin, a relative lack of hair, and the ease with which it can be isolated from respiratory movements31. Indeed, the ear dermis has been used to image the interstitial behavior of dendritic cells32,33, T cells28,29,34,35, and neutrophils36,37, and is a well-established site for studying dermal inflammation. Increasingly, non-invasive procedures have been replacing surgical preparations of the skin, including split dermis38,39, flank39,40, or dorsal skin flap window39,41 models, that can induce changes to the local inflammatory milieu. The use of transferred, in vitro-primed, antigen-specific CD4 effector T cells allows for the study of a homogenous population of cells in the context of a dermal inflammatory response30. Here we describe a non-invasive imaging procedure that allows for the visualization of antigen-specific effector CD4 T cells in the dermal interstitium of the inflamed mouse ear, and the ability to manipulate these cells in real-time by introducing blocking antibodies through a venous catheter. We show that this model is effective for tracking the movement of CD4 T cells in the dermis and for querying the mechanisms that govern this motility.

<table><tbody><tr><td>BALB/c mice</td><td>Jackson Laboratories</td><td>000651</td><td>Mice used were bred in-house</td></tr><tr><td>DO11.10 mice</td><td>Jackson Laboratories</td><td>003303</td><td>Mice used were bred in-house</td></tr><tr><td>HBSS</td><td>Fisher</td><td>10-013-CV</td><td>Multiple Equivalent</td></tr><tr><td>Newborn Calf Serum (NCS)</td><td>Thermo/HyClone</td><td>SH30118.03</td><td>Heat inactivated at 56 &amp;deg;C for 30 minutes</td></tr><tr><td>Guinea Pig Complement</td><td>Cedarlane</td><td>CL-5000</td><td></td></tr><tr><td>anti-CD8 antibody</td><td>ATCC</td><td>3.155 (ATCC TIB-211)</td><td>Antibodies derived from&amp;nbsp; this hybridoma</td></tr><tr><td>anti-MHC Class II antibody</td><td>ATCC</td><td>M5/114.15.2 (ATCC TIB-120)</td><td>Antibodies derived from&amp;nbsp; this hybridoma</td></tr><tr><td>anti-CD24 antibody</td><td>ATCC</td><td>J11d.2 (ATCC TIB-183)</td><td>Antibodies derived from&amp;nbsp; this hybridoma</td></tr><tr><td>anti-Thy1.2 antibody</td><td>ATCC</td><td>J1j.10 (ATCC TIB-184)</td><td>Antibodies derived from&amp;nbsp; this hybridoma</td></tr><tr><td>Ficoll (Fico/Lite-LM)</td><td>Atlanta Biologicals</td><td>I40650</td><td></td></tr><tr><td>PBS</td><td>Fisher</td><td>21-040-CV</td><td>Multiple Equivalent</td></tr><tr><td>EDTA</td><td>Fisher</td><td>15323591</td><td></td></tr><tr><td>biotinylated anti-CD62L antibody (clone MEL-14)</td><td>BD</td><td>553149</td><td></td></tr><tr><td>streptavidin magnetic separation beads</td><td>Miltenyi</td><td>130-048-101</td><td></td></tr><tr><td>MACS LS Separation Column</td><td>Miltenyi</td><td>130-042-401</td><td></td></tr><tr><td>recombinant human IL-2</td><td>Peprotech</td><td>200-02</td><td></td></tr><tr><td>recombinant mouse IL-4</td><td>Peprotech</td><td>214-14</td><td></td></tr><tr><td>recombinant mouse IL-12</td><td>Peprotech</td><td>210-12</td><td></td></tr><tr><td>anti-IFNg antibody (clone XMG 1.2)</td><td>eBioscience</td><td>16-7311-85</td><td></td></tr><tr><td>anti-IL-4 antibody (clone 11b11)</td><td>eBioscience</td><td>16-7041-85</td><td></td></tr><tr><td>RPMI</td><td>VWR</td><td>45000-412</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Fisher</td><td>15303641</td><td></td></tr><tr><td>L-glutamine</td><td>Fisher</td><td>15323671</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>Bio-Rad</td><td>161-0710</td><td></td></tr><tr><td>ovalbumin peptide</td><td>Biopeptide</td><td></td><td>ISQAVHAAHAEINEAGR-OH peptide</td></tr><tr><td>Fetal Calf Serum (FCS)</td><td>Thermo/HyClone</td><td>SV30014.03</td><td>Heat inactivated at 56 &amp;deg;C for 30 minutes</td></tr><tr><td>24-well culture plate</td><td>LPS</td><td>3526</td><td>Multiple Equivalent</td></tr><tr><td>CFSE</td><td>Life Technologies</td><td>C34554</td><td></td></tr><tr><td>CMTMR</td><td>Life Technologies</td><td>C2927</td><td></td></tr><tr><td>28 G1/2 insulin syringes, 1ml</td><td>BD</td><td>329420</td><td></td></tr><tr><td>28 G1/2 insulin syringes, 300&amp;mu;l</td><td>BD</td><td>309301</td><td></td></tr><tr><td>27 G1/2 TB syringes, 1ml</td><td>BD</td><td>309623</td><td></td></tr><tr><td>30 G1/2 needles</td><td>BD</td><td>305106</td><td></td></tr><tr><td>PE-10 medical tubing</td><td>BD</td><td>427400</td><td></td></tr><tr><td>cyanoacrylate veterinary adhesive (Vetbond)</td><td>3M</td><td>1469SB</td><td></td></tr><tr><td>heating plate</td><td>WPI</td><td>61830</td><td></td></tr><tr><td>Heating plate controller</td><td>WPI</td><td>ATC-2000</td><td></td></tr><tr><td>Water blanket controller</td><td>Gaymar</td><td>TP500</td><td>No longer in production, newer equivalent available</td></tr><tr><td>water blanket</td><td>Kent Scientific</td><td>TP3E</td><td></td></tr><tr><td>Isoflurane vaporizer</td><td>LEI Medical</td><td>Isotec 4</td><td>No longer in production, newer equivalent available</td></tr><tr><td>isoflurane</td><td>Henry Schein</td><td></td><td>Ordered through Veterinary staff</td></tr><tr><td>microcentrifuge tubes</td><td>VWR</td><td>20170-038</td><td>Multiple Equivalent</td></tr><tr><td>medical tape</td><td>3M</td><td>1538-0</td><td></td></tr><tr><td>isoflurane nosecone</td><td></td><td></td><td>Built In-house, see Fig 2</td></tr><tr><td>imaging platform</td><td></td><td></td><td>Built In-house, see Fig 2</td></tr><tr><td>curved forceps</td><td>WPI</td><td>15915-G</td><td>Multiple Equivalent</td></tr><tr><td>scissors</td><td>Roboz</td><td>RS-6802</td><td>Multiple Equivalent</td></tr><tr><td>glass coverslips</td><td>VWR</td><td></td><td>Multiple Equivalent</td></tr><tr><td>high vacuum grease</td><td>Fisher</td><td>146355D</td><td></td></tr><tr><td>cotton swabs</td><td></td><td></td><td>Multiple Equivalent</td></tr><tr><td>delicate task wipes</td><td>Fisher</td><td>34155</td><td>Multiple Equivalent</td></tr><tr><td>Olympus Fluoview 1000 AOM-MPM upright microscope with Spectra-Physics MaiTai HP DeepSee Ti:Sa laser</td><td>Olympus</td><td></td><td>call for quote</td></tr><tr><td>optical table with vibration control</td><td>Newport</td><td></td><td>call for quote</td></tr><tr><td>25x NA 1.05 water immersion objective for multiphoton imaging</td><td>Olympus</td><td>XLPLN25XWMP2</td><td></td></tr><tr><td>objective heater</td><td>Bioptechs</td><td>PN 150815</td><td></td></tr><tr><td>Detection filter cube</td><td>Olympus</td><td>FV10-MRVGR/XR</td><td>Proprietary cube, can be approximated from individual filters/dichroics</td></tr><tr><td>anti-integrin &amp;beta;1 antibody (clone hMb1-1)</td><td>eBioscience</td><td>16-0291-85</td><td>Azide free, low endotoxin</td></tr><tr><td>anti-integrin &amp;beta;3 antibody (clone 2C9.G3)</td><td>eBioscience</td><td>16-0611-82</td><td>Azide free, low endotoxin</td></tr><tr><td>Texas Red Dextran (70,000 MW)</td><td>Life Technologies</td><td>D-1830</td><td></td></tr></tbody></table>

imaging cd4 t cell interstitial migration in the inflamed dermis

CD4 + T细胞进行的效应子功能的能力取决于通过一个尚未未定义机制这些细胞在发炎外周组织的迅速和有效的迁移。多光子显微镜对免疫系统的研究中的应用提供了测量的完整组织内的免疫应答的动力学的工具。这里，我们提出在发炎的小鼠耳真皮的CD4 T细胞的非侵入性活体多光子成像的协议。使用定制成像平台和静脉导管允许的CD4 T细胞动力学在真皮间质的可视化，通过加入阻断抗体中涉及能动性关键分子组分来询问这些细胞中的实时的能力。该系统提供了在体外模型和微创手术成像程序既有优势。了解的CD4 T细胞的活力所使用的途径，最终可能提供洞察到基CD4 + T细胞的C函数以及自身免疫性疾病的慢性感染的发病机制和病理。

研究原位免疫反应，而不改变免疫环境的能力是研究与发炎组织T效应细胞的实时交互必需的。由这个协议中，在图1A和B中概述的完好耳真皮的成像，允许转移荧光标记的T效应细胞在皮肤间质的可视化。这允许两个高分辨率（ 图1C）和延时（ 图1D， 电影1）在发炎的真皮效应T细胞动力学的图片。 <p cl...

Watch this Scientific Journal Video about Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis at JoVE.com

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

成像的CD4 T细胞间质迁移发炎真皮

支配CD4效应T细胞的间质动力在炎症部位的机制比较陌生。我们提出了一种非侵入性的方式来可视化和体外 -primed CD4T细胞操纵在发炎耳朵真皮，允许这些细胞在原位的动态行为的研究。

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

imaging-cd4-t-cell-interstitial-migration-in-the-inflamed-dermis

Research

JoVE Journal

Immunology and Infection

10.6K 次观看.  University of Rochester School of Medicine and Dentistry, Rochester, NY. 支配CD4效应T细胞的间质动力在炎症部位的机制比较陌生。我们提出了一种非侵入性的方式来可视化和体外 -primed CD4T细胞操纵在发炎耳朵真皮，允许这些细胞在原位的动态行为的研究。 

视频: Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the tumor microenvironment and respond adequately to tumor antigens. The recent development of improved imaging approaches, such as 2-photon microscopy, and the use of powerful mouse tumor models have shed light on some of the mechanisms that regulate anti-tumor T cell activities. Whereas such systems have provided valuable insights, they do not always predict human responses. In human, our knowledge in the field mainly comes from a description of fixed tumor samples from human patients, as well as in vitro studies. However, in vitro models lack the complex three-dimensional tumor milieu and, therefore, are incomplete approximations of in vivo T cell activities. Fresh slices made from explanted tissue represent a complex multi-cellular tumor environment that can act as an important link between co-cultured studies and animal models. Originally set up in murine lymph nodes1 and previously described in a JoVE article2, this approach has now been transposed to human tumors to examine the dynamics of both plated3&#160;as well as resident&#160;T cells4.
Here, a protocol for the preparation of human lung tumor slices, immunostaining of resident CD8 T and tumor cells, and tracking of CD8 T lymphocytes within the tumor microenvironment using confocal microscopy is described. This system is uniquely placed to screen for novel immunotherapy agents favoring T cell migration in tumors.

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

This protocol describes a method to image resident tumor-infiltrating CD8 T cells labeled with fluorescently coupled antibodies within human lung tumor slices. This technique permits real-time analyses of CD8 T cell migration using confocal microscopy.

Ex 活体人肺肿瘤切片中居民 CD8 T 淋巴细胞的共聚焦显微成像

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the ...

科研

癌症研究

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.

生发中心的高度已解决活体条纹照明显微镜

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

免疫与感染

In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers

This report demonstrates the use of major histocompatibility complex (MHC) class II dextramers for detection of autoreactive CD4 T cells in situ in myelin proteolipid protein (PLP) 139-151-induced experimental autoimmune encephalomyelitis (EAE) in SJL mice and cardiac myosin heavy chain-&#945; (Myhc) 334-352-induced experimental autoimmune myocarditis (EAM) in A/J mice. Two sets of cocktails of dextramer reagents were used, where dextramers+ cells were analyzed by laser scanning confocal microscope (LSCM): EAE, IAs/PLP 139-151 dextramers (specific)/anti-CD4 and IAs/Theiler&#8217;s murine encephalomyelitis virus (TMEV) 70-86 dextramers (control)/anti-CD4; and EAM, IAk/Myhc 334-352 dextramers/anti-CD4 and IAk/bovine ribonuclease (RNase) 43-56 dextramers (control)/anti-CD4. LSCM analysis of brain sections obtained from EAE mice showed the presence of cells positive for CD4 and PLP 139-151 dextramers, but not TMEV 70-86 dextramers suggesting that the staining obtained with PLP 139-151 dextramers was specific. Likewise, heart sections prepared from EAM mice also revealed the presence of Myhc 334-352, but not RNase 43-56-dextramer+ cells as expected. Further, a comprehensive method has also been devised to quantitatively analyze the frequencies of antigen-specific CD4 T cells in the &#8216;Z&#8217; serial images.

In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers

The protocol to detect self-reactive CD4 T cells in brain and heart by direct staining with major histocompatibility complex class II dextramers has been described in this report. For comprehensive analysis, a reliable method to enumerate the frequencies of antigen-specific CD4+ T cells in situ is also devised.

原位检测自身反应性CD4 + T细胞在大脑和心脏使用主要组织相容性复合II类Dextramers的

This report demonstrates the use of major histocompatibility complex (MHC) class II dextramers for detection of autoreactive CD4 T cells in situ in myelin ...

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring.
The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange.
To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4).
CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a detailed insight into their altered behavior upon liver injury and therefore their potential involvement in disease progression.

Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

免疫细胞健康和疾病肝脏使用CXCR6.Gfp记者小鼠长期活体多光子显微镜成像

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological synapses&#39;. Within these intimate cell-cell interfaces discrete sub-cellular clusters of MHC/Ag-TCR, F-actin, adhesion and signaling molecules form and remodel rapidly. These dynamics are thought to be critical determinants of both the efficiency and quality of the immune responses that develop and therefore of protective versus pathologic immunity. Current understanding of immunological synapses with physiologic APCs is limited by the inadequacy of the obtainable imaging resolution. Though artificial substrate models (e.g., planar lipid bilayers) offer excellent resolution and have been extremely valuable tools, they are inherently non-physiologic and oversimplified. Vascular and lymphatic endothelial cells have emerged as an important peripheral tissue (or stromal) compartment of &#39;semi-professional APCs&#39;. These APCs (which express most of the molecular machinery of professional APCs) have the unique feature of forming virtually planar cell surface and are readily transfectable (e.g., with fluorescent protein reporters). Herein a basic approach to implement endothelial cells as a novel and physiologic &#39;planar cellular APC model&#39; for improved imaging and interrogation of fundamental antigenic signaling processes will be described.

Adaptive immunity is controlled by dynamic 'immunological synapses' formed between T cells and antigen presenting cells. This protocol describes methods for investigating endothelial cells both as understudied physiologic APCs and as a novel type of 'planar cellular APC model'.

成像免疫突触动力学内皮平面细胞模型

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological ...

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph node (DLN). The mobilization of DCs to the DLN is complex and remains to be fully elucidated during infection. Herein described is the use of an innovative, simple assay that relies on the fluorochrome 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) to track the migration of DCs during footpad infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin (BCG) in C57BL/6 mice. This assay enables the characterization of skin DC sub-populations that actively relocate to the draining, popliteal LN in response to BCG. This protocol originates from a BCG model where migratory skin DCs were identified by flow cytometry. The assay is amiable to the study and identification of DCs or other cells that home to the popliteal LN after inoculation of microbes, their metabolites or other inflammatory stimuli in the footpad, and consequently to study factors that regulate the migration of these cells.

An assay in mice to track cell migration from skin to draining lymph node is described which enables the characterization of skin Dendritic cells mobilized to the lymph node after footpad infection with Bacille Calmette-Gu&#233;rin.

基于CFSE-试验鼠皮肤树突状细胞的迁移习进引流淋巴结在与感染<em&gt;牛分枝杆菌</em&gt;卡介苗

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph ...

Light-sheet Microscopy for Three-dimensional Visualization of Human Immune Cells

In vivo, activation, proliferation, and function of immune cells all occur in a three-dimensional (3D) environment, for instance in lymph nodes or tissues. Up to date, most in vitro systems rely on two-dimensional (2D) surfaces, such as cell-culture plates or coverslips. To optimally mimic physiological conditions in vitro, we utilize a simple 3D collagen matrix. Collagen is one of the major components of extracellular matrix (ECM) and has been widely used to constitute 3D matrices. For 3D imaging, the recently developed light-sheet microscopy technology (also referred to as single plane illumination microscopy) is featured with high acquisition speed, large penetration depth, low bleaching, and photocytotoxicity. Furthermore, light-sheet microscopy is particularly advantageous for long-term measurement. Here we describe an optimized protocol how to set up and handle human immune cells, e.g. primary human cytotoxic T lymphocytes (CTL) and natural killer (NK) cells in the 3D collagen matrix for usage with the light-sheet microscopy for live cell imaging and fixed samples. The procedure for image acquisition and analysis of cell migration are presented. A particular focus is given to highlight critical steps and factors for sample preparation and data analysis. This protocol can be employed for other types of suspension cells in a 3D collagen matrix and is not limited to immune cells.

Here, we present a protocol to visualize immune cells embedded in a three-dimensional (3D) collagen matrix using light-sheet microscopy. This protocol also elaborates how to track cell migration in 3D. This protocol can be employed for other types of suspension cells in the 3D matrix.

三维人体免疫细胞可视化的光片显微术

In vivo, activation, proliferation, and function of immune cells all occur in a three-dimensional (3D) environment, for instance in lymph nodes or ...

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.

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;

毛里小肠内皮淋巴细胞的内质成像

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

Advanced Imaging of Lung Homing Human Lymphocytes in an Experimental In Vivo Model of Allergic Inflammation Based on Light-sheet Microscopy

Overwhelming tissue accumulation of highly activated immune cells represents a hallmark of various chronic inflammatory diseases and emerged as an attractive therapeutic target in the clinical management of affected patients. In order to further optimize strategies aiming at therapeutic regulation of pathologically imbalanced tissue infiltration of pro-inflammatory immune cells, it will be of particular importance to achieve improved insights into disease- and organ-specific homing properties of peripheral lymphocytes. The here described experimental protocol allows to monitor lung accumulation of fluorescently labeled and adoptively transferred human lymphocytes in the context of papain-induced pulmonary inflammation. In contrast to standard in vitro assays frequently used for the analysis of immune cell migration and chemotaxis, the now introduced in vivo setting takes into account lung-specific aspects of tissue organization and the influence of the complex inflammatory scenario taking place in the living murine organism. Moreover, three-dimensional cross-sectional light-sheet fluorescence microscopic imaging does not only provide quantitative data on infiltrating immune cells, but also depicts the pattern of immune cell localization within the inflamed lung. Overall, we are able to introduce an innovative technique of high value for immunological research in the field of chronic inflammatory lung diseases, which can be easily applied by following the provided step-by-step protocol.

The here introduced protocol allows characterization of the lung homing capacity of primary human lymphocytes under in vivo inflammatory conditions. Pulmonary infiltration of adoptively transferred human immune cells in a mouse model of allergic inflammation can be imaged and quantified by light-sheet fluorescence microscopy of chemically cleared lung tissue.

基于光片显微镜的变应性炎症体内模型中肺呼名人淋巴细胞的高级成像

Overwhelming tissue accumulation of highly activated immune cells represents a hallmark of various chronic inflammatory diseases and emerged as an ...

医学

Quantifying Leukocyte Egress via Lymphatic Vessels from Murine Skin and Tumors

Leukocyte egress from peripheral tissues to draining lymph nodes is not only critical for immune surveillance and initiation but also contributes to the resolution of peripheral tissue responses. While a variety of methods are used to quantify leukocyte egress from non-lymphoid, peripheral tissues, the cellular and molecular mechanisms that govern context-dependent egress remain poorly understood. Here, we describe the use of in situ photoconversion for quantitative analysis of leukocyte egress from murine skin and tumors. Photoconversion allows for the direct labeling of leukocytes resident within cutaneous tissue. Though skin exposure to violet light induces local inflammatory responses characterized by leukocyte infiltrates and vascular leakiness, in a head-to-head comparison with transdermal application of fluorescent tracers, photoconversion specifically labeled migratory dendritic cell populations and simultaneously enabled the quantification of myeloid and lymphoid egress from cutaneous microenvironments and tumors. The mechanisms of leukocyte egress remain a missing component in our understanding of intratumoral leukocyte complexity, and thus the application of the tools described herein will provide unique insight into the dynamics of tumor immune microenvironments both at steady state and in response to therapy.

Here, we demonstrate the methods for in vivo quantification of leukocyte egress from na&#239;ve, inflamed, and malignant murine skin. We perform a head-to-head comparison of two models: transdermal FITC application and in situ photoconversion. Furthermore, we demonstrate the utility of photoconversion for tracking leukocyte egress from cutaneous tumors.

小鼠皮肤和肿瘤淋巴血管白细胞出口的定量

Leukocyte egress from peripheral tissues to draining lymph nodes is not only critical for immune surveillance and initiation but also contributes to the ...