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露真皮或皮下组织，该协议阻止损伤诱导的炎症和嗜中性粒细胞37和其他免疫细胞的快速招募进入真皮。在本系统中使用静脉导管的递送阻断抗体对关键分子允许DYNA的实时询问CD4 + T细胞的MIC行为。使用这种成像协议已经揭示了在体外系统8时没有发现的CD4 T细胞间质活力30的关键要求。 在该过程的关键步骤 在任何成像协议的一个重要步骤是确保用于成像的稳定组织制备。重要的是要确保有耳朵和盖玻片之间有足够的PBS存在任何气泡和耳是在与玻璃接触，但没有那么多，以使该磁带变得从玻璃脱粘。同样，避免了带保持盖玻片到平台和任何真空润滑脂将防止磁带松动随时间之间的接触。温度也必须保持恒定，以避免振荡和从热收缩或平台材料的膨胀漂移。 限制和修改 45为活时间推移成像的模型。然而，耳朵的常住免疫人口是从皮肤上侧面或足垫46不同，并且当相对于其他部位47的小鼠耳具有明显的血管特性。因此，对于一些应用来说，耳朵的成像数据到其他皮肤部位的比较可能是困难的。 该协议还要求出血流并进入真皮转移的T效应细胞的有效迁移。这限制使用具有在归巢或外渗缺陷细胞，因为它们将不能进入间隙空间的能力。外渗可通过注入细胞直接进入耳真皮37,48，尽管这会造成这种方式可能无法概括在体内外渗经历细胞的定位或行为的一些机械损伤和细胞的递送被绕过。 也考虑使用未着色的受体小鼠进行成像实验，如这里或白化的C57BL / 6- 酪氨酸 的c-2J小鼠中使用的BALB / c小鼠品系的关键。在色素小鼠黑色素，除了是高度自发荧光，下从多光子激光37即使是相对低功率的激发加热。这可能会导致对皮肤和随后的炎症36或荧光斑点31，复杂化的结果的热损伤。这可能限制了可在多参数成像实验中使用的荧光团。然而，一些非常明亮或高表达的荧光分子可以在较低的激光功率有效激发，所有由于在色素小鼠有效的可视化。 未来的应用 因为这是一种非侵入性的过程，它可以很容易地适于与在延长的时间周期序列成像的小鼠的纵向研究。而这里描述会随着时间的推移周期褪色大于3-4天荧光标记，使用内源荧光的细胞或荧光报道细胞的消除了这个问题。事实上，我们先前使用这个协议来跟踪接近体内产生的抗原特异性效应轴承细胞因子的记者，包括IFNγ记者雪人30,49和IL-4记者4get 50。我们还可视化的内源性CD4细胞CD4-Cre重组ROSA26一站式两侧装接loxP EYFP荧光记者小鼠30。作为EYFP和其他荧光蛋白质的荧光性能，从化学染料如CFSE不同，可能需要修改的摄像参数高效的可视化。如增加像素停留时间可提高有些暗淡的荧光团的荧光信号，并减少时间推移图像的长度可以减轻可能观察到的任何漂白变化。在900 nm激发，我们以前没有观察到CFSE，CMTMR或EYFP的显著漂白过短的成像时间间隔。 虽然此过程关注测量CD4效应T细胞运动的动态，但并不限于这种应用。工作目前正在进行中，以测量与抗原通过使用荧光报道小鼠和荧光标记的抗体的注射入真皮呈递细胞标记的细胞或于成像51,52之前组织结构的效应T细胞的动态相互作用。此外，虽然此协议演示了如何使用导管提供封闭抗体，而成像，其他化合物，包括小分子抑制剂，能给药。最终，该协议提供了一个灵活的平台随时间来测量免疫动力学， 体内 ，以非侵入性方式。

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 border="0" cellpadding="0" cellspacing="0" width="1060">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">BALB/c mice</td>
			<td style="width:145px;">Jackson Laboratories</td>
			<td style="width:196px;">000651</td>
			<td style="width:472px;">Mice used were bred in-house</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">DO11.10 mice</td>
			<td>Jackson Laboratories</td>
			<td>003303</td>
			<td>Mice used were bred in-house</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">HBSS</td>
			<td>Fisher</td>
			<td>10-013-CV</td>
			<td>Multiple Equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Newborn Calf Serum (NCS)</td>
			<td>Thermo/HyClone</td>
			<td>SH30118.03</td>
			<td>Heat inactivated at 56 &#176;C for 30 minutes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Guinea Pig Complement</td>
			<td>Cedarlane</td>
			<td>CL-5000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">anti-CD8 antibody</td>
			<td>ATCC</td>
			<td>3.155 (ATCC TIB-211)</td>
			<td>Antibodies derived from&#160; this hybridoma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">anti-MHC Class II antibody</td>
			<td>ATCC</td>
			<td>M5/114.15.2 (ATCC TIB-120)</td>
			<td>Antibodies derived from&#160; this hybridoma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">anti-CD24 antibody</td>
			<td>ATCC</td>
			<td>J11d.2 (ATCC TIB-183)</td>
			<td>Antibodies derived from&#160; this hybridoma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">anti-Thy1.2 antibody</td>
			<td>ATCC</td>
			<td>J1j.10 (ATCC TIB-184)</td>
			<td>Antibodies derived from&#160; this hybridoma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Ficoll (Fico/Lite-LM)</td>
			<td>Atlanta Biologicals</td>
			<td>I40650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">PBS</td>
			<td>Fisher</td>
			<td>21-040-CV</td>
			<td>Multiple Equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">EDTA</td>
			<td>Fisher</td>
			<td>15323591</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">biotinylated anti-CD62L antibody (clone MEL-14)</td>
			<td>BD</td>
			<td>553149</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">streptavidin magnetic separation beads</td>
			<td>Miltenyi</td>
			<td>130-048-101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">MACS LS Separation Column</td>
			<td>Miltenyi</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">recombinant human IL-2</td>
			<td>Peprotech</td>
			<td>200-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">recombinant mouse IL-4</td>
			<td>Peprotech</td>
			<td>214-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">recombinant mouse IL-12</td>
			<td>Peprotech</td>
			<td>210-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">anti-IFNg antibody (clone XMG 1.2)</td>
			<td>eBioscience</td>
			<td>16-7311-85</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">anti-IL-4 antibody (clone 11b11)</td>
			<td>eBioscience</td>
			<td>16-7041-85</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">RPMI</td>
			<td>VWR</td>
			<td>45000-412</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Penicillin/Streptomycin</td>
			<td>Fisher</td>
			<td>15303641</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">L-glutamine</td>
			<td>Fisher</td>
			<td>15323671</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">2-mercaptoethanol</td>
			<td>Bio-Rad</td>
			<td>161-0710</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">ovalbumin peptide</td>
			<td>Biopeptide</td>
			<td></td>
			<td>ISQAVHAAHAEINEAGR-OH peptide</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Fetal Calf Serum (FCS)</td>
			<td>Thermo/HyClone</td>
			<td>SV30014.03</td>
			<td>Heat inactivated at 56 &#176;C for 30 minutes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">24-well culture plate</td>
			<td>LPS</td>
			<td>3526</td>
			<td>Multiple Equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">CFSE</td>
			<td>Life Technologies</td>
			<td>C34554</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">CMTMR</td>
			<td>Life Technologies</td>
			<td>C2927</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">28 G1/2 insulin syringes, 1ml</td>
			<td>BD</td>
			<td>329420</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">28 G1/2 insulin syringes, 300&#956;l</td>
			<td>BD</td>
			<td>309301</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">27 G1/2 TB syringes, 1ml</td>
			<td>BD</td>
			<td>309623</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">30 G1/2 needles</td>
			<td>BD</td>
			<td>305106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">PE-10 medical tubing</td>
			<td>BD</td>
			<td>427400</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">cyanoacrylate veterinary adhesive (Vetbond)</td>
			<td>3M</td>
			<td>1469SB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">heating plate</td>
			<td>WPI</td>
			<td>61830</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Heating plate controller</td>
			<td>WPI</td>
			<td>ATC-2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Water blanket controller</td>
			<td>Gaymar</td>
			<td>TP500</td>
			<td>No longer in production, newer equivalent available</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">water blanket</td>
			<td>Kent Scientific</td>
			<td>TP3E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Isoflurane vaporizer</td>
			<td>LEI Medical</td>
			<td>Isotec 4</td>
			<td>No longer in production, newer equivalent available</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">isoflurane</td>
			<td>Henry Schein</td>
			<td></td>
			<td>Ordered through Veterinary staff</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">microcentrifuge tubes</td>
			<td>VWR</td>
			<td>20170-038</td>
			<td>Multiple Equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">medical tape</td>
			<td>3M</td>
			<td>1538-0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">isoflurane nosecone</td>
			<td></td>
			<td></td>
			<td>Built In-house, see Fig 2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">imaging platform</td>
			<td></td>
			<td></td>
			<td>Built In-house, see Fig 2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">curved forceps</td>
			<td>WPI</td>
			<td>15915-G</td>
			<td>Multiple Equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">scissors</td>
			<td>Roboz</td>
			<td>RS-6802</td>
			<td>Multiple Equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">glass coverslips</td>
			<td>VWR</td>
			<td></td>
			<td>Multiple Equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">high vacuum grease</td>
			<td>Fisher</td>
			<td>146355D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">cotton swabs</td>
			<td></td>
			<td></td>
			<td>Multiple Equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">delicate task wipes</td>
			<td>Fisher</td>
			<td>34155</td>
			<td>Multiple Equivalent</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:247px;">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 height="21">
			<td height="21" style="height:21px;width:247px;">optical table with vibration control</td>
			<td>Newport</td>
			<td></td>
			<td>call for quote</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">25x NA 1.05 water immersion objective for multiphoton imaging</td>
			<td>Olympus</td>
			<td>XLPLN25XWMP2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">objective heater</td>
			<td>Bioptechs</td>
			<td>PN 150815</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">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 height="42">
			<td height="42" style="height:42px;width:247px;">anti-integrin &#946;1 antibody (clone hMb1-1)</td>
			<td>eBioscience</td>
			<td>16-0291-85</td>
			<td>Azide free, low endotoxin</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">anti-integrin &#946;3 antibody (clone 2C9.G3)</td>
			<td>eBioscience</td>
			<td>16-0611-82</td>
			<td>Azide free, low endotoxin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">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。 <img alt="图1" src="/files/ftp_upload/53585/53585fig1.jpg" /> 图1.高分辨率和完整的真皮间质T效应细胞的4D成像 。 （A）试验大纲。耳prepar的（b）照片编用于与乳液的位点（黑虚线）和最佳区域用于成像（红线）成像指示。从纤维状胶原（蓝色）和德克萨斯红-葡聚糖表示CFSE标记的Th1细胞（绿色）高分辨率栈，第二谐波信号（C）的最大3D投影标记的脉管（红色）。白色箭头指示的几个自身荧光毛囊之一。标尺代表50微米（D）跟踪在CFA发炎真皮30分钟Th1细胞迁徙路径。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53585/53585fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 该成像协议要求使用在内部构造的专门成像平台，以及一个适于鼻锥为吸入麻醉而成像的交付。成像平台（ 图2A-B）由Øf相一升高的中心部的铝底板。此凸起部具有与丙烯酸衬里的插入部感觉提供给耳支撑而不压缩和潜在破坏性的薄组织。我们设置的几何形状需要灵活可调鼻锥的建设成像过程中，提供吸入麻醉。此鼻锥，包括连接到柔性管（ 图2C）的50毫米部分的改性微量离心管的，被固定在适当位置具有修饰的离心管组成的保持器（ 图2D），并通过钩和环紧固件固定到成像平台。使用钩和环紧固件的允许鼻锥组件，使得小鼠的任一耳可以成像的重新定位，并且可以最佳地定位成用于单个小鼠。 <img alt="图2" src="/files/ftp_upload/53585/53585fig2.jpg" /> 图2.球菌pment的活体成像。定制的成像平台的顶部（A）和侧面（B）的意见。鼻锥异氟醚管理（C）和鼻锥座（D）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53585/53585fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 尾静脉插管允许连续访问循环施用抗体和其它小分子可扩散出来的脉管的，或如高分子葡聚糖较大的荧光分子标记的血管。抗β1和抗β3整经导管封闭抗体，真皮内以前运动细胞阻滞（ 电影2）为100微克给药后。这些细胞具有抗体后降低平均速度<html>施用（ 图3A），以及在曲折指数显著减少，总位移的总轨道长度（ 图3B）的比率。 <img alt="图3" src="/files/ftp_upload/53585/53585fig3.jpg" /> 图3. 抗β1 和抗β3 抗体 的施用 抑制在CFA中发炎的皮肤Th1细胞迁移。（A）的Th1细胞的平均速度之前和100μg抗β1和抗β3整给药后封闭抗体。 （B）的Th1细胞之前和抗体阻断后的蜿蜒索引。大约100跟踪细胞来自图像之前和在一个代表性的实验一个鼠标抗体阻断后。由曼惠特尼统计。ES / ftp_upload / 53585 / 53585fig3large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 因为头发不会从耳的表面除去，从头发成像伪影是常见的。应避免从毛囊（ 图4A），并从上覆毛发（ 图4B）阴影和自发荧光自身荧光在可能，因为它们可以掩盖的T细胞和用自动图像分析软件干扰。同样，夹在耳表面与盖玻片之间的气泡可导致成像伪影（ 图4C）。耳的适当的准备，应尽量减少任何剩余气泡​​的数量和大小。 <img alt="图4" src="/files/ftp_upload/53585/53585fig4.jpg" /> 图4。 从头发中自发荧光及耳前差的公共构件paration（一）自体荧光毛囊。 （B）的自体荧光毛发和毛囊（绿色）和胶原（白色）与上覆头发阴影，从而导致在图像中的暗线。从气泡（C）的工件，示出了位移，自体荧光角化表皮的边缘（虚线）。标尺代表50微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53585/53585fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 另外，使用的加热多个源可导致与组织的稳定性由于热膨胀和收缩的问题。不正确恒温器的设置，例如，可以成像，可以使结果困难（ 电影3）的解释期间导致大振荡。重要的是要确定该提供恒定的温度和最大化稳定性的任何系统的最佳设置。 Ultimat伊利，温度受控成像室是由于温度变化而除去变性的最佳方式。 <img alt="电影1" src="/files/ftp_upload/53585/53585movie1.jpg" /> <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53585/gaylomovie1.mov" target="_blank">电影1 Th1细胞在CFA发炎真皮迁移（右键点击下载）。</a> 30分钟时间推移图像显示Th1细胞（绿色）的真皮层胶原蛋白网络的迁移（二次谐波产生，蓝色）。 <img alt="电影2" src="/files/ftp_upload/53585/53585movie2.jpg" /> <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53585/gaylomovie2.mov" target="_blank">电影2 Th1细胞停滞在1β阻滞和β3整合（右键点击下载）。</a>细胞（绿色）被拍摄30分钟的ADMINIST前阻断抗体的定量，然后成像在同一位置的另一20分钟。 <img alt="电影3" src="/files/ftp_upload/53585/53585movie3.jpg" /> <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53585/gaylomovie3.mov" target="_blank">电影3.从控制不佳的加热板振荡（右键点击下载）。</a> Th1细胞（绿色）和二次谐波产生（蓝色）的30分钟时间推移图像。这个图像被收集后，所使用的加热板被发现有一个错误的恒温器，在成像的平台和随后的热膨胀和收缩的温度引起的振荡。

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

Nanyang Technological University

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

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

T细胞受体在小鼠T细胞的逆转录病毒转导

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

科研

免疫与感染

Competitive Homing Assays to Study Gut-tropic T Cell Migration

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial cells and tissue extravasation is a multistep process controlled by different adhesion molecules (homing receptors) expressed on lymphocytes and their respective ligands (addressins) displayed on endothelial cells 1 2. Even though the function of these adhesion receptors can be partially studied ex vivo, the ultimate test for their physiological relevance is to assess their role during in vivo lymphocyte adhesion and migration. Two complementary strategies have been used for this purpose: intravital microscopy (IVM) and homing experiments. Although IVM has been essential to define the precise contribution of specific adhesion receptors during the adhesion cascade in real time and in different tissues, IVM is time consuming and labor intensive, it often requires the development of sophisticated surgical techniques, it needs prior isolation of homogeneous cell populations and it permits the analysis of only one tissue/organ at any given time. By contrast, competitive homing experiments allow the direct and simultaneous comparison in the migration of two (or even more) cell subsets in the same mouse and they also permit the analysis of many tissues and of a high number of cells in the same experiment.
Here we describe the classical competitive homing protocol used to determine the advantage/disadvantage of a given cell type to home to specific tissues as compared to a control cell population. We chose to illustrate the migratory properties of gut-tropic versus non gut-tropic T cells, because the intestinal mucosa is the largest body surface in contact with the external environment and it is also the extra-lymphoid tissue with the best-defined migratory requirements. Moreover, recent work has determined that the vitamin A metabolite all-trans retinoic acid (RA) is the main molecular mechanism responsible for inducing gut-specific adhesion receptors (integrin a4b7and chemokine receptor CCR9) on lymphocytes. Thus, we can readily generate large numbers of gut-tropic and non gut-tropic lymphocytes ex vivoby activating T cells in the presence or absence of RA, respectively, which can be finally used in the competitive homing experiments described here.

Competitive homing experiments allow to directly assessing the migratory properties of two different cell populations in a single mouse. Here we illustrate this procedure by comparing the migration of ex vivo-generated gut-tropic versus non-gut tropic T cells.

竞争的原点实验研究肠嗜T细胞迁移

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial ...

A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular organisms towards a source of nutrients or away from unsuitable conditions, as well as in multicellular organisms for tissue development, immune surveillance and wound healing, just to mention a few roles1,2,3. Deregulation of this process can lead to serious neurological, cardiovascular and immunological diseases, as well as exacerbated tumor formation and spread4,5. Molecularly, actin polymerization and receptor recycling have been shown to play important roles in creating cellular extensions (lamellipodia), that drive the forward movement of the cell6,7,8. However, many biological questions about cell migration remain unanswered.
The central role for cellular motility in human health and disease underlines the importance of understanding the specific mechanisms involved in this process and makes accurate methods for evaluating cell motility particularly important. Microscopes are usually used to visualize the movement of cells. However, cells move rather slowly, making the quantitative measurement of cell migration a resource-consuming process requiring expensive cameras and software to create quantitative time-lapsed movies of motile cells. Therefore, the ability to perform a quantitative measurement of cell migration that is cost-effective, non-laborious, and that utilizes common laboratory equipment is a great need for many researchers.
The phagokinetic track motility assay utilizes the ability of a moving cell to clear gold particles from its path to create a measurable track on a colloidal gold-coated glass coverslip9,10. With the use of freely available software, multiple tracks can be evaluated for each treatment to accomplish statistical requirements. The assay can be utilized to assess motility of many cell types, such as cancer cells11,12, fibroblasts9, neutrophils13, skeletal muscle cells14, keratinocytes15, trophoblasts16, endothelial cells17, and monocytes10,18-22. The protocol involves the creation of slides coated with gold nanoparticles (Au&deg;) that are generated by a reduction of chloroauric acid (Au3+) by sodium citrate. This method was developed by Turkevich et al. in 195123 and then improved in the 1970s by Frens et al.24,25. As a result of this chemical reduction step, gold particles (10-20 nm in diameter) precipitate from the reaction mixture and can be applied to glass coverslips, which are then ready for use in cellular migration analyses9,26,27.
In general, the phagokinetic track motility assay is a quick, quantitative and easy measure of cellular motility. In addition, it can be utilized as a simple high-throughput assay, for use with cell types that are not amenable to time-lapsed imaging, as well as other uses depending on the needs of the researcher. Together, the ability to quantitatively measure cellular motility of multiple cell types without the need for expensive microscopes and software, along with the use of common laboratory equipment and chemicals, make the phagokinetic track motility assay a solid choice for scientists with an interest in understanding cellular motility.

The phagokinetic motility track assay is a method used to assess the movement of cells. Specifically, the assay measures chemokinesis (random cell motility) over time in a quantitative manner. The assay takes advantage of the ability of cells to create a measurable track of their movement on colloidal gold-coated coverslips.

细胞迁移的的Phagokinetic轨道运动MLCK的定量评价

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular ...

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.
Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

T细胞实时成像实时信号复合物的形成

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

监测树突状细胞迁移<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H磁共振成像

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

隔离和幼稚的CD4 T细胞的Th17细胞分化

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects.
Here, we depict an in vitro experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10R&#945; and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 &#176;C, 5% CO2. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

We developed an in vitro assay to investigate the role of immunoregulatory pathways in the regulation of cytokine secretion by HIV-specific CD4 T cells.

在体外试验 ，以评估免疫调节途径对HIV特异性CD4 + T细胞效应功能的影响

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. 
In general, both CD4+ and CD8+ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-Abm12) in the antigen-presenting groove of the MHC-class II (I-Ab) molecule is sufficient to induce strong allogeneic CD4+ T cell activation in C57BL/6 mice. Skin grafts from I-Abm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3-/- and Rag2-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 104 wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-&#947; in I-Abm12-transplanted Rag2-/- mice.

Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

尾部皮肤来研究异基因CD4 + T细胞应答的小鼠移植

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

诱导小鼠肠道炎症由效应CD4的过继转移<sup&gt; +</sup&gt; CD45RB<sup&gt;高</sup&gt; T细胞免疫缺陷小鼠

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

鼠标朴素CD4<sup&gt; +</sup&gt; T细胞分离和<em&gt;在体外</em&gt;分化为T细胞亚群

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...