Name: in vivo photolabeling of cells in the colon to assess migratory potential of hematopoietic cells in neonatal mice
Uploaded: 2018-08-10T14:45:00
Description: Watch this Scientific Journal Video about In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice at JoVE.com

尼亚耆那得到了 NIH/NIAID 职业转型奖1K22AI116661-01 的支持。

所有动物程序都是在马萨诸塞州总医院的机构动物护理和使用委员会 (IACUC) 批准和遵守的情况下进行的。
注意: 此协议涉及使用3b 类激光 (LG3)。LG3 激光安全护目镜在操作激光时必须始终使用。必须遵循适当的培训和安全准则, 以避免受伤的危险。
1. 激光的设计与装配
<ol><li>通过连接到 led 的4针 M8 连接器电缆, 将发光二极管 (LED) (405 nm、19.3 兆瓦、1...

<ol>
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细胞的识别和鉴定与结肠菌群的影响是重要的, 应该有助于理解如何将信息从粘膜微环境传递到身体的其余部分。研究肠道相关细胞迁移的一种方法, 需要将肠道相关细胞分离出来, 然后将移植到受体小鼠体内, 以确定它们的组织自导模式和功能12,13。此方法是有限的, 因为仅可以跟踪传输的特定单元格类型。此外, 由于从组织中分离细胞和纯化过程而产生...

哺乳动物胃肠道有数以百计的细菌存在于与宿主1的共生关系中。在当地环境中存在的免疫细胞加强了与这些微生物的和平共处, 并对病菌入侵建立了保护性屏障。因此, 免疫细胞和微生物群之间的双向相互作用是建立一个共生社区, 教育宿主免疫系统并设定免疫反应性的阈值的关键。微生物组成的变化, 或 dysbiosis, 可能扰乱免疫动态平衡和扰乱调节电路, 抑制肠道炎症导致免疫介导的疾病, 如1型糖尿病和 IBD2,3.
出生后的时间是一个独特的发展窗口, 肠道微生物群落开始建立的同时免疫系统成熟4。产后微生物群是不稳定的, 与转移在社区构成自然地和频繁地发生5。与微生物群相互作用的免疫细胞位于肠道的两个不同的解剖位置--固有层和肠道上皮6。许多类型的免疫细胞存在于肠道, 包括淋巴细胞 (如 T 细胞, B 细胞, 和先天淋巴细胞) 以及髓细胞 (包括树突状细胞, 单核, 和巨细胞)。这些细胞, 也称为造血细胞, 执行多种功能, 保持肠道屏障和维持动态平衡。
除了在肠道部位的调节功能外, 粘膜的免疫细胞还可能携带微生物信息到肠外的部位, 以调节全身免疫7、8、9。这是一个日益增长的研究兴趣的领域, 并强调需要的方法, 以确定免疫细胞迁移出肠道组织, 以探讨其功能。这里报告的协议使用了一个商业上可用的老鼠模型, 其中 photoconvertible 荧光蛋白被利用来标记细胞。范切除小鼠无所不在表达绿色荧光 Dendra2 蛋白, 是不可逆转地切换到红色荧光激活时紫外线 (UV) 光10。利用光纤套管将405纳米光传送到新生小鼠结肠中, 我们证明了 photoconverted 造血细胞 (起源于或通过结肠过渡) 可以在脾脏中找到。

<table border="0" cellpadding="0" cellspacing="0" style="width:873px;" width="874">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:873px;">Laser</td>
			<td style="width:201px;"></td>
			<td style="width:167px;"></td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:259px;">Light Emitting Diode (LED)</td>
			<td style="width:201px;">THORLABS</td>
			<td style="width:167px;">M405FP1</td>
			<td style="width:247px;">CAUTION: this is a Class 3b laser. Safety goggles must be worn when using the laser. It emits a 405 nm wavelength with a current of 1400 mA. It is fiber-coupled. It accepts SMA connector. https://www.thorlabs.com/thorproduct.cfm?partnumber=M405FP1</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:259px;">LED driver</td>
			<td style="width:201px;">THORLABS</td>
			<td style="width:167px;">LEDD1B</td>
			<td style="width:247px;">Drives a constant current of 1200 mA through the laser. https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=2616</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:259px;">Optogenetics patch cable</td>
			<td style="width:201px;">THORLABS</td>
			<td style="width:167px;">M87L01</td>
			<td style="width:247px;">1 m long cable with an SMA connector. https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=11405&#38;pn=M87L01#11454</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:259px;">Fiber optic cannula</td>
			<td style="width:201px;">Doric lenses&#160;</td>
			<td style="width:167px;">MFC_480/500-0.5_5mm_ZF1.25_C45</td>
			<td style="width:247px;">5 mm long cannula with an outer diameter of 500 &#181;m and an inner diameter of 480 &#181;m. The NA value is 0.5. The ferrule is zirconia, 1.25 mm OD. https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=6036</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:259px;">Power supply</td>
			<td style="width:201px;">THORLABS</td>
			<td style="width:167px;">KPS101</td>
			<td style="width:247px;">Supplies 15 V with a current of 2.4 A https://www.thorlabs.com/search/thorsearch.cfm?search=KPS101</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:259px;">LG3 laser safety goggles&#160;</td>
			<td style="width:201px;">THORLABS</td>
			<td style="width:167px;">LG3</td>
			<td style="width:247px;">Orange lenses with 47% visible light transmission https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=762&#38;pn=LG3#2523</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">Red light&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>74327-10</td>
			<td style="width:247px;">15 W lamp https://us.vwr.com/store/product/12360027/paterson-safelight-electron-microscopy-sciences</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:873px;">Intestinal cell isolation</td>
			<td style="width:201px;"></td>
			<td style="width:167px;"></td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:259px;">Isoflurane</td>
			<td style="width:201px;">Patterson Veterinary</td>
			<td style="width:167px;">07-893-1389</td>
			<td style="width:247px;">CAUTION: inhalation of this anesthetic may cause dizziness, drowsiness, or even unconsciousness. This anesthetic should be used in a Class II hood.&#160; https://www.pattersonvet.com/Supplies/ProductFamilyDetails/PIF_762328?carouselPageNumber=3</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:259px;">1X HBSS</td>
			<td style="width:201px;">Gibco</td>
			<td style="width:167px;">14025076</td>
			<td style="width:247px;">Ca/Mg free https://www.fishersci.com/shop/products/gibco-hbss-calcium-magnesium-no-phenol-red-4/14025076?searchHijack=true&#38;searchTerm=14025076&#38;searchType=RAPID&#38;matchedCatNo=14025076</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Calf Serum</td>
			<td style="width:201px;">Hyclone AZM</td>
			<td style="width:167px;">197696</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:259px;">EDTA</td>
			<td style="width:201px;">Invitrogen</td>
			<td style="width:167px;">15575020</td>
			<td style="width:247px;">0.5 M concentration https://www.thermofisher.com/order/catalog/product/15575020?SID=srch-srp-15575020</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:259px;">DTT</td>
			<td style="width:201px;">Sigma</td>
			<td style="width:167px;">10197777001</td>
			<td style="width:247px;">CAUTION: harmful if swallowed and causes skin irritation. 1 M concentration https://www.sigmaaldrich.com/catalog/product/roche/dttro?lang=en&#38;region=US</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:259px;">HEPES</td>
			<td style="width:201px;">Gibco</td>
			<td style="width:167px;">15630080</td>
			<td style="width:247px;">1 M concentration https://www.thermofisher.com/order/catalog/product/15630080?SID=srch-hj-15630080</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:259px;">Petri dish</td>
			<td style="width:201px;">Corning</td>
			<td style="width:167px;">353004</td>
			<td style="width:247px;">https://www.fishersci.com/shop/products/falcon-easy-grip-tissue-culture-dishes-2/08772f?searchHijack=true&#38;searchTerm=08772F&#38;searchType=RAPID&#38;matchedCatNo=08772F</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:259px;">70 micron cell strainer</td>
			<td style="width:201px;">Falcon</td>
			<td style="width:167px;">352350</td>
			<td style="width:247px;">https://www.fishersci.com/shop/products/falcon-cell-strainers-4/087712</td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:259px;">Micro magnetic stir bar</td>
			<td style="width:201px;">Fisherbrand</td>
			<td style="width:167px;">1451364</td>
			<td style="width:247px;">Rinse in 70% ethanol after each use. Rinse several times in distilled water prior to each use. The bar is 8 mm long with an octagonal shape. https://www.fishersci.com/shop/products/fisherbrand-octagonal-magnetic-stir-bars-12/1451364#?keyword=1451364</td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:259px;">Magnetic stir plate</td>
			<td style="width:201px;">Corning Laboratory Stirrers</td>
			<td style="width:167px;">440826</td>
			<td style="width:247px;">https://www.coleparmer.com/i/corning-440826-nine-position-stirrer-120-vac-60-hz/8430420?PubID=UX&#38;persist=true&#38;ip=no&#38;gclid=CjwKCAiAqbvTBRAPEiwANEkyCLPLrWABXmOUI0QE53NLV0Owxlcs2V1K6rWbRPOwlcVVDq000FBiQxoCqQAQAvD_BwE</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:259px;">Collagenase</td>
			<td style="width:201px;">Roche</td>
			<td style="width:167px;">5401020001</td>
			<td style="width:247px;">https://www.sigmaaldrich.com/catalog/product/roche/05401020001?lang=en&#38;region=US&#38;gclid=CjwKCAiAjuPRBRBxEiwAeQ2QPhE44qlvxjmo1PYu3zCas3w-_d6P9gKjXW82-c1EOm6NjPHCc5WuixoC_0IQAvD_BwE</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:259px;">DNase I</td>
			<td style="width:201px;">Sigma</td>
			<td style="width:167px;">10104159001</td>
			<td style="width:247px;">https://www.sigmaaldrich.com/catalog/product/roche/10104159001?lang=en&#38;region=US</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:259px;">1X PBS</td>
			<td style="width:201px;">Gibco</td>
			<td style="width:167px;">20012-027</td>
			<td style="width:247px;">https://www.thermofisher.com/order/catalog/product/20012027?SID=srch-hj-20012-027</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:259px;">Pipet aid</td>
			<td style="width:201px;">Thermo Scientific</td>
			<td style="width:167px;">14387165</td>
			<td style="width:247px;">https://www.fishersci.com/shop/products/s1-pipette-fillers/14387165#?keyword=14387165</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:259px;">10 mL serological pipet</td>
			<td style="width:201px;">Falcon</td>
			<td style="width:167px;">357530</td>
			<td style="width:247px;">https://www.fishersci.com/shop/products/falcon-serological-pipets-bulk-pack-5/p-163659</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:259px;">25 mL serological pipet</td>
			<td style="width:201px;">Falcon</td>
			<td style="width:167px;">357515</td>
			<td style="width:247px;">https://www.fishersci.com/shop/products/falcon-serological-pipets-bulk-pack-5/p-163659</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:259px;">15 mL conical centrifuge tube</td>
			<td style="width:201px;">Thermo Scientific</td>
			<td style="width:167px;">339651</td>
			<td style="width:247px;">https://www.thermofisher.com/order/catalog/product/339650</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:259px;">50 mL conical centrifuge tube</td>
			<td style="width:201px;">Thermo Scientific</td>
			<td style="width:167px;">339653</td>
			<td style="width:247px;">https://www.thermofisher.com/order/catalog/product/339650</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:873px;">Single cell suspension</td>
			<td style="width:201px;"></td>
			<td style="width:167px;"></td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:259px;">Eppendorf tubes</td>
			<td style="width:201px;">Seal-Rite</td>
			<td style="width:167px;">1615-5500</td>
			<td style="width:247px;">Holds 1.5 mL. https://www.usascientific.com/Seal-Rite-1.5-ml-tube.aspx</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:259px;">Tissue homogenizer&#160;</td>
			<td style="width:201px;">Kimble</td>
			<td>K7495400000</td>
			<td style="width:247px;">Requires 2 AA batteries. https://www.fishersci.com/shop/products/kontes-pellet-pestle-cordless-motor-cordless-motor/k7495400000</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:259px;">Homogenizer tips&#160;</td>
			<td style="width:201px;">Kimble</td>
			<td style="width:167px;">7495210590</td>
			<td style="width:247px;">Plastic, 0.5 mL tips https://www.fishersci.com/shop/products/kimble-chase-kontes-pellet-pestle-14/k7495210590#?keyword=7495210590</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:259px;">ACK lysing buffer</td>
			<td style="width:201px;">Gibco</td>
			<td style="width:167px;">A10492-01</td>
			<td style="width:247px;">https://www.thermofisher.com/order/catalog/product/A1049201?SID=srch-hj-A10492-01</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:259px;">40 micron cell strainer</td>
			<td style="width:201px;">Falcon</td>
			<td style="width:167px;">08-771-1</td>
			<td style="width:247px;">https://www.fishersci.com/shop/products/falcon-cell-strainers-4/087711</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:873px;">Antibodies</td>
			<td style="width:201px;"></td>
			<td style="width:167px;"></td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:259px;">BV786 anti-mouse CD45&#160;</td>
			<td style="width:201px;">BD</td>
			<td style="width:167px;">564225</td>
			<td style="width:247px;">Clone 3O-F11 https://www.bdbiosciences.com/us/reagents/research/antibodies-buffers/immunology-reagents/anti-mouse-antibodies/cell-surface-antigens/bv786-rat-anti-mouse-cd45-30-f11/p/564225</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Live/Dead</td>
			<td>Invitrogen</td>
			<td>L34962</td>
			<td style="width:247px;">https://www.thermofisher.com/order/catalog/product/L34962</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Other</td>
			<td style="width:201px;"></td>
			<td style="width:167px;"></td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Razor blades</td>
			<td>VWR</td>
			<td>55411-050</td>
			<td style="width:247px;">Use for decapitation. https://us.vwr.com/store/product/4548306/vwr-razor-blades</td>
		</tr>
	</tbody>
</table>

in vivo photolabeling of cells in the colon to assess migratory potential of hematopoietic cells in neonatal mice

肠道细菌群落是早期建立的, 对免疫细胞的发育和功能有影响。新生儿微生物群易受多种外部影响, 包括抗生素的使用和饮食, 这会影响对自身免疫和炎症性疾病的敏感性。炎症性肠病 (IBD) 等疾病的特点是大量的免疫细胞涌入肠道。然而, 由微生物群所调节的免疫细胞可能会另外从肠道中移出, 以影响肠道的免疫应答。因此, 有必要识别和鉴定可能携带微生物信息从肠道到远端部位的细胞。在这里, 我们描述了一种方法来标记细胞在结肠的新生小鼠在体内, 使他们的身份, 在肠外的地方, 迁移后鉴定。

用光纤电缆将405纳米光传送到2天的老范切除小鼠的冒号中。在以前的实验中, 三十年代的暴露被确定为最大 photoconversion 的结肠细胞具有最小的细胞毒性 (图 1a)。因此, 按照《议定书》的规定, 对结肠的不同部分进行连续三十年代的曝光。激光照射后, 老鼠立即被安乐死, 细胞的 photoconversion 被确定。采用荧光共轭 anti-CD45 抗体对全?...

Watch this Scientific Journal Video about In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice at JoVE.com

In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice

这里描述的协议使用了 photolabeling 方法在新生老鼠特别辨认免疫细胞移居从结肠到肠外的站点。这一策略将有助于研究早期生命中宿主-微生物的相互作用。

体内结肠细胞 Photolabeling 对新生小鼠造血细胞迁移潜能的评价

Enteric bacterial communities are established early in life and influence immune cell development and function. The neonatal microbiota is susceptible to ...

Research

JoVE Journal

Medicine

In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice

Making Sense of Listening: The IMAP Test Battery

听力：IMAP测试电池的决策意识

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the ...

科研

医学

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

一项战略，以确定在常见的疾病，如自闭症和精神分裂症的新突变

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

颈动脉输液的紫杉烷类化合物在小鼠体内的药代动力学和药效学分析

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

宫内遥测来衡量鼠标收缩压力<em&gt;在体内</em

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

在锝-99m在马腱末端标记的骨髓间充质干细胞的体内成像和跟踪

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

研究胰腺癌干细胞特性开发新的治疗策略

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Injection of Syngeneic Murine Melanoma Cells to Determine Their Metastatic Potential in the Lungs

同基因小鼠黑色素瘤细胞注射，以确定其在肺转移潜能

Approximately 90% of human cancer deaths are linked to metastasis.  Despite the prevalence and relative harm of metastasis, therapeutics for treatment or ...

Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration

心肌梗死新生小鼠，心肌再生的模型

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

从人类肿瘤异种移植的小鼠细胞的消耗显着改善靶细胞下游分析

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

体外和体内检测人类细胞中的 Mitophagy,线虫和小鼠

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...