Name: observing islet function and islet-immune cell interactions in live pancreatic tissue slices
Uploaded: 2021-04-12T13:30:00
Description: Watch this Scientific Journal Video about Observing Islet Function and Islet-Immune Cell Interactions in Live Pancreatic Tissue Slices at JoVE.com

这项工作由NIH拨款R01 DK123292，T32 DK108736，UC4 DK104194，UG3 DK122638和P01 AI042288资助。这项研究是在糖尿病胰腺器官捐献者网络（nPOD;RRID：SCR_014641），由JDRF（nPOD：5-SRA-2018-557-Q-R）和Leona M. & Harry B. Helmsley慈善信托基金（Grant #2018PG-T1D053）赞助的1型糖尿病合作研究项目。所表达的内容和观点是作者的责任，并不一定反映nPOD的官方观点。与nPOD合作提供研究资源的器官采购组织（OPO）列在 http://www.jdrfnpod.org/for-partners/npod-partners/。感谢佛罗里达大学的Kevin Otto博士提供用于生成小鼠切片的振动切片机。

注意：所有使用小鼠的实验方案均已获得佛罗里达大学动物护理和使用委员会（201808642）的批准。来自两性组织供体的人类胰腺切片是通过佛罗里达大学糖尿病胰腺器官供体网络（nPOD）组织库获得的。根据器官捐赠法律法规，与nPOD合作的认证器官采购组织从尸体器官捐献者身上摘取了人类胰腺，并被佛罗里达大学机构审查委员会（IRB No. 392-2008）归类为"非人类受试者"，免除了同意的需要。专门?...

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该协议的目的是解释胰腺切片的产生以及在功能和免疫学研究中使用切片所需的程序。使用活胰腺切片有很多好处。然而，有几个关键步骤对于组织在所描述的实验方案期间保持活力和有用至关重要。必须快速工作。应尽量减少注射胰腺和在振动切片机上产生切片之间的时间长度，以保持组织的活力。通过在切片之前将胰腺保持在冷ECS中，而不是室温ECS，还可以提高生存能力。重要的是，切片不?...

胰腺受累是胰腺炎、T1D 和 T2D1，2，3 等疾病的特征性特征。孤立胰岛功能的研究通常涉及将胰岛从周围环境中移除4。开发了活胰腺组织切片方法，以允许研究胰腺组织，同时保持完整的胰岛微环境并避免使用应激的胰岛分离程序5，6，7。来自人类供体组织的胰腺组织切片已被成功用于研究T1D，并且除了免疫细胞浸润之外，还显示出β细胞丢失和功能障碍的过程8，9，10，11，12，13。活胰腺组织切片法可应用于小鼠和人胰腺组织5，6，8。来自器官捐献者组织的人类胰腺组织切片是通过与糖尿病胰腺器官捐献者网络（nPOD）合作获得的。小鼠切片可以从各种不同的小鼠品系中生成。
该协议将侧重于非肥胖糖尿病重组激活基因-1-null（NOD.Rag1-/-） 和 T 细胞受体转基因 （AI4） （NOD.Rag1-/-.AI4 α/β）小鼠品系。点头。Rag1-/- 小鼠由于重组激活基因1（Rag1）14的破坏而无法发育T细胞和B细胞。点头。Rag1-/-.AI4 α/β 小鼠被用作加速型1型糖尿病的模型，因为它们产生靶向胰岛素表位的单个T细胞克隆，导致胰岛持续浸润和快速疾病发展15。此处的方案描述了通过应用共聚焦显微镜方法使用活人胰腺切片进行功能和免疫学研究的程序。本文描述的技术包括活力评估，胰岛鉴定和定位，胞质Ca2 + 记录，以及免疫细胞群的染色和鉴定。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>#3 Style Scalpel Handle</td>
			<td>Fisherbrand</td>
			<td>12-000-163</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M HEPES</td>
			<td>Fisher Scientific</td>
			<td>BP299-100</td>
			<td>HEPES Buffer, 1M Solution</td>
		</tr>
		<tr>
			<td>10 cm Untreated Culture Dish</td>
			<td>Corning</td>
			<td>430591</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Luer-Lok Syringe</td>
			<td>BD</td>
			<td>301029</td>
			<td>BD Syringe with Luer-Lok Tips</td>
		</tr>
		<tr>
			<td>27 G Needle</td>
			<td>BD</td>
			<td>BD 305109</td>
			<td>BD General Use and PrecisionGlide Hypodermic Needles</td>
		</tr>
		<tr>
			<td>35 mm coverglass-bottom Petri dish</td>
			<td>Ibidi</td>
			<td>81156</td>
			<td>&#181;-Dish 35 mm, high</td>
		</tr>
		<tr>
			<td>50 mL syringe</td>
			<td>BD</td>
			<td>309653</td>
			<td></td>
		</tr>
		<tr>
			<td>8-well chambered coverglass</td>
			<td>Ibidi</td>
			<td>80826</td>
			<td>&#181;-Slide 8 Well</td>
		</tr>
		<tr>
			<td>APC anti-mouse CD8a antibody</td>
			<td>Biolegend</td>
			<td>100712</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Fisher Scientific</td>
			<td>199898</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Sigma</td>
			<td>C5670</td>
			<td>CaCl2</td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>Sigma</td>
			<td>C7902</td>
			<td>CaCl2 (dihydrate)</td>
		</tr>
		<tr>
			<td>Compact Digital Rocker</td>
			<td>Thermo Fisher Scientific</td>
			<td>88880020</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal laser-scanning microscope</td>
			<td>Leica</td>
			<td>SP8</td>
			<td>Pinhole&#8201;= 1.5-2 airy units; acquired with 10x/0.40 numerical aperture HC PL APO CS2 dry and 20x/0.75 numerical aperture HC PL APO CS2 dry objectives at 512&#8201;&#215;&#8201;512 pixel resolution</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma</td>
			<td>G7021</td>
			<td>C6H12O6</td>
		</tr>
		<tr>
			<td>ddiH2O</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dithizone</td>
			<td>Sigma-Aldrich</td>
			<td>D5130-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Invitrogen</td>
			<td>D12345</td>
			<td>Dimethyl sulfoxide</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Decon Laboratories</td>
			<td>2805</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 35 mm tissue culture dish</td>
			<td>Corning</td>
			<td>353001</td>
			<td>Falcon Easy-Grip Tissue Culture Dishes</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>10082147</td>
			<td></td>
		</tr>
		<tr>
			<td>Feather No. 10 Surgical Blade</td>
			<td>Electron Microscopy Sciences</td>
			<td>7204410</td>
			<td></td>
		</tr>
		<tr>
			<td>fluo-4-AM</td>
			<td>Invitrogen</td>
			<td>F14201</td>
			<td>cell-permeable Ca2+ indicator</td>
		</tr>
		<tr>
			<td>Gel Control Super Glue</td>
			<td>Loctite</td>
			<td>45198</td>
			<td></td>
		</tr>
		<tr>
			<td>Graefe Forceps</td>
			<td>Fine Science Tools</td>
			<td>11049-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Hardened Fine Scissors</td>
			<td>Fine Science Tools</td>
			<td>14090-09</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Gibco</td>
			<td>14025092</td>
			<td>Hanks Balanced Salt Solution</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H4034</td>
			<td>C8H18N2O4S</td>
		</tr>
		<tr>
			<td>Ice bucket</td>
			<td>Fisherbrand</td>
			<td>03-395-150</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Patterson Veterinary</td>
			<td>NDC 14043-704-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Johns Hopkins Bulldog Clamp</td>
			<td>Roboz Surgical Store</td>
			<td>RS-7440</td>
			<td>&#160;Straight; 500-900 Grams Pressure; 1.5&#34; Length</td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Kimberly-Clark Professional</td>
			<td>34705</td>
			<td>Kimtech Science&#8482; Kimwipes&#8482; Delicate Task Wipers, 2-Ply</td>
		</tr>
		<tr>
			<td>LIVE/DEAD Viability/Cytotoxicity Kit</td>
			<td>Invitrogen</td>
			<td>L3224</td>
			<td>This kit contains the calcein-AM live cell dye.</td>
		</tr>
		<tr>
			<td>Low glucose DMEM</td>
			<td>Corning</td>
			<td>10-014-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride hexahydrate</td>
			<td>Sigma</td>
			<td>M9272</td>
			<td>MgCl2 (hexahydrate)</td>
		</tr>
		<tr>
			<td>Magnesium sulfate heptahydrate</td>
			<td>Sigma</td>
			<td>M2773</td>
			<td>MgSO4 (heptahydrate)</td>
		</tr>
		<tr>
			<td>Magnetic Heated Platform</td>
			<td>Warner Instruments</td>
			<td>PM-1</td>
			<td>Platform for imaging chamber for dynamic stimulation recordings</td>
		</tr>
		<tr>
			<td>Microwave</td>
			<td>GE</td>
			<td>JES1460DSWW</td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene Syringe Filter</td>
			<td>Thermo Fisher Scientific</td>
			<td>726-2520</td>
			<td></td>
		</tr>
		<tr>
			<td>No.4 Paintbrush</td>
			<td>Michaels</td>
			<td>10269140</td>
			<td></td>
		</tr>
		<tr>
			<td>Open Diamond Bath Imaging Chamber</td>
			<td>Warner Instruments</td>
			<td>RC-26</td>
			<td>Imaging chamber for dynamic stimulation recordings</td>
		</tr>
		<tr>
			<td>Oregon Green 488 BAPTA-1-AM</td>
			<td>Invitrogen</td>
			<td>O6807</td>
			<td>cell-permeable Ca2+ indicator</td>
		</tr>
		<tr>
			<td>Overnight imaging chamber</td>
			<td>Okolab</td>
			<td>H201-LG</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Thermo Fisher Scientific</td>
			<td>20012050</td>
			<td>To make agarose for slice generation</td>
		</tr>
		<tr>
			<td>PE-labeled insulin tetramer</td>
			<td>Emory Tetramer Research Core</td>
			<td>sequence YAIENYLEL</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma</td>
			<td>P5405</td>
			<td>KCl</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma</td>
			<td>P5655</td>
			<td>KH2PO4</td>
		</tr>
		<tr>
			<td>Razor Blades</td>
			<td>Electron Microscopy Sciences</td>
			<td>71998</td>
			<td>For Vibratome; Double Edge Stainless Steel, uncoated</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Gibco</td>
			<td>11875093</td>
			<td></td>
		</tr>
		<tr>
			<td>SeaPlaque low melting-point agarose</td>
			<td>Lonza</td>
			<td>50101</td>
			<td>To make agarose for slice generation</td>
		</tr>
		<tr>
			<td>Slice anchor</td>
			<td>Warner Instruments</td>
			<td>64-1421</td>
			<td></td>
		</tr>
		<tr>
			<td>Slice anchor (dynamic imaging)</td>
			<td>Warner Instruments</td>
			<td>640253</td>
			<td>Slice anchor for dynamic imaging chamber</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td>NaHCO3</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma</td>
			<td>S5886</td>
			<td>NaCl</td>
		</tr>
		<tr>
			<td>Sodium phosphate monohydrate</td>
			<td>Sigma</td>
			<td>S9638</td>
			<td>NaH2PO4 (monohydrate)</td>
		</tr>
		<tr>
			<td>Soybean Trypsin Inhibitor</td>
			<td>Sigma</td>
			<td>T6522-1G</td>
			<td>Trypsin inhibitor from Glycine max (soybean)</td>
		</tr>
		<tr>
			<td>Stage Adapter</td>
			<td>Warner Instruments</td>
			<td>SA-20MW-AL</td>
			<td>To fit imaging chamber for dynamic stimulation recordings on the microscope stage</td>
		</tr>
		<tr>
			<td>Stage-top incubator</td>
			<td>Okolab</td>
			<td>H201</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereoscope</td>
			<td>Leica</td>
			<td>IC90 E MSV266</td>
			<td></td>
		</tr>
		<tr>
			<td>SYTOX Blue Dead Cell Stain</td>
			<td>Invitrogen</td>
			<td>S34857</td>
			<td>blue-fluorescent nucleic acid stain</td>
		</tr>
		<tr>
			<td>Transfer Pipet</td>
			<td>Falcon</td>
			<td>357575</td>
			<td>Falcon&#8482; Plastic Disposable Transfer Pipets</td>
		</tr>
		<tr>
			<td>Valve Control System</td>
			<td>Warner Instruments</td>
			<td>VCS-8</td>
			<td>System for dynamic stimulation recordings</td>
		</tr>
		<tr>
			<td>Vibratome VT1000 S</td>
			<td>Leica</td>
			<td>VT1000 S</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Fisher Scientific</td>
			<td>FSGPD02</td>
			<td>Fisherbrand Isotemp General Purpose Deluxe Water Bath GPD 02</td>
		</tr>
	</tbody>
</table>

observing islet function and islet-immune cell interactions in live pancreatic tissue slices

活的胰腺组织切片允许在完整的胰岛微环境中研究胰岛的生理学和功能。切片由嵌入琼脂糖中的活人和小鼠胰腺组织制备，并使用振动切片机切割。这种方法允许组织保持活力和功能，除了保留潜在的病理，如1型（T1D）和2型糖尿病（T2D）。切片方法通过维持构成胰腺内分泌和外分泌组织的复杂结构和各种细胞间相互作用，为胰腺的研究提供了新的方向。该协议展示了如何对胰腺切片内的活内源性免疫细胞进行染色和延时显微镜检查，以及胰岛生理学评估。此外，可以使用主要的组织相容性复合物多聚体试剂来改进这种方法，以识别胰岛细胞抗原特异性的免疫细胞群。

该协议将产生适合功能研究和免疫细胞记录的活胰腺组织切片。明场和反射光下的切片外观如图 1A，B所示。如前所述，胰岛可以使用反射光在切片中找到，因为它们的颗粒度增加，由于其胰岛素含量而发生（图1C），并且在使用反射光时与背景组织相比被清楚地观察到。应在切片生成后评估生存能力，如果超过20%的胰岛不可行，则不应记?...

Watch this Scientific Journal Video about Observing Islet Function and Islet-Immune Cell Interactions in Live Pancreatic Tissue Slices at JoVE.com

Observing Islet Function and Islet-Immune Cell Interactions in Live Pancreatic Tissue Slices

本研究介绍了活胰腺组织切片在胰岛生理学和胰岛免疫细胞相互作用研究中的应用。

观察活胰腺组织切片中的胰岛功能和胰岛-免疫细胞相互作用

Live pancreatic tissue slices allow for the study of islet physiology and function in the context of an intact islet microenvironment. Slices are prepared ...

我们的方案将有助于回答免疫细胞相互作用对胰岛功能的影响。活胰腺组织切片方法允许研究胰岛的生理学和功能，同时保持潜在的病理和天然微环境。这种方法可以提供对胰腺疾病的疾病过程的见解，例如胰腺炎，I型糖尿病和II型糖尿病。首先，将块排列在试样夹具座上，使其不超过刀片宽度，确保当振动器缓慢移动时，刀片可以向前移动尽可能短的距离。在试样支架上涂上一行超级胶水，使用胶水分配器的末端形成薄层，然后将组织块翻转到胶水上，使靠近组织的一面朝上。轻轻按下块，让胶水干燥三分钟。接下来，用螺钉将板连接到振动器上，并调整刀片高度和行进距离，以便刀片在块的长度上移动，并且勉强高于它们。用镊子轻轻轻轻推块以检查胶水是否干燥，然后用冷却的细胞外溶液填充振动盘，直到刀片被覆盖。将振动切片机设置为120微米厚的切片并启动它，然后观察从组织块上脱落的切片。当切片从块上漂浮时，将画笔或镊子放在它们下面，然后将切片放在含有三毫摩尔D-葡萄糖和胰蛋白酶抑制剂的Krebs-Ringer碳酸氢盐缓冲液中。将含有切片的板放在摇臂上，并在室温下以25 RPM孵育一小时。如果切片需要保存更长时间，请将切片置于15毫升切片培养基中，并将平板置于培养箱中。将准备好的切片在37摄氏度下孵育以进行当天研究，或在实验前将24摄氏度培养过夜的切片转移到37摄氏度至少一小时。在记录前至少一小时打开显微镜，并将载物台顶部培养箱平衡至37摄氏度。将盖板玻璃底培养皿固定在舞台上，其中包含切片。通过在明场模式下设置10X物镜来聚焦显微镜，并通过在切片中寻找橙棕色的椭圆形来使用明场模式定位小岛。通过按下显微镜触摸屏控制器上的CS按钮，将显微镜切换到共聚焦成像。要使用反射率查看胰岛，请打开 638 激光探测器和 PMT 探测器。将激光功率设置为1%至2%，然后关闭陷波滤波器。使用405纳米激光和PMT检测器观察核酸染色。混合检测器用于CD8抗体检测和488纳米激光和混合检测器，用于观察胰岛素四聚体。使用载物台控制器的 X 和 Y 旋钮将感兴趣的小岛居中置于视野中。找到感兴趣的小岛后，切换到20倍物镜并放大，使小岛占据大部分帧。取一个z-stack的胰岛，然后找到细胞活着的最佳光学切片，并且任何周围的免疫细胞都处于焦点中。将显微镜设置为以XYZT模式记录，并优化设置以在几个小时内每20分钟记录一次所选步骤的z-stack。使用明场和反射光显微镜观察胰腺组织切片中的胰岛。胰岛中的胰岛素增加了颗粒度并导致大量反射光的吸收，增加了胰岛的可见性。通过染色评估活人胰腺组织切片的活力。活细胞以绿色表示，死细胞以蓝色表示。生存能力的另一个积极指标是可观察到的基础活性。当活的胰腺切片加载细胞可渗透的钙指示剂时，在小鼠和人胰腺切片中观察到葡萄糖刺激。还量化了葡萄糖刺激期间单个细胞的荧光。使用地噻嗪染色和反射光显微镜观察完整和患病的胰岛。由于免疫细胞浸润和细胞死亡，患病胰岛开始失去颗粒。免疫细胞群可以使用CD8抗体和胰岛素四聚体染色来鉴定。共染色表明这些细胞是特异性靶向胰岛素抗原的效应性T细胞。在此过程中要记住的最关键的事情是始终将切片保留在溶液蛋白酶抑制剂中。遵循该程序，可以执行免疫细胞实验中的许多功能，从而允许研究免疫细胞相互作用和胰岛功能的影响。

Research

JoVE Journal

Immunology and Infection

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

动态活细胞成像的宿主 - 病原体相互作用的研究使用的光学陷阱

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

科研

免疫与感染

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

单细胞测量液泡破裂引起细胞内病原体

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed ...

Visualizing Protein-DNA Interactions in Live Bacterial Cells Using Photoactivated Single-molecule Tracking

可视化的活细菌细胞蛋白-DNA相互作用使用光活化的单分子跟踪

Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome ...

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

一个<em&gt;体外</em&gt;急性脑片的少突胶质细胞定向T细胞攻击模式

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

RNA的疗效和毒性的评价基因或药物治疗靶向HIV-1的生产使用

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

精密切割的小鼠肺切片可视化现场肺树突状细胞

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for ...

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

体内人淋巴组织和女性生殖器粘膜与人体免疫机能丧失病毒1和 Histoculture 的感染

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under ...

Electrochemiluminescence Assays for Human Islet Autoantibodies

人胰岛自身抗体的电化学发光测定

Pinpointing islet autoantibodies associated with type 1 diabetes (T1D) leads the way to project and deter this disease in the general population. A novel ...

A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals

小鼠胰岛细胞筛选致糖尿病环境化学物质的研究

Exposure to certain environmental chemicals in human and animals has been found to cause cellular damage of the pancreatic &#946; cells which will lead to ...

Determination of Regulatory T Cell Subsets in Murine Thymus, Pancreatic Draining Lymph Node and Spleen Using Flow Cytometry

流式细胞术测定小鼠胸腺、胰腺干淋巴结和脾脏的调节 t 细胞亚群

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, ...