这项工作是由美国国立卫生研究院（R01HL128714到CF）和（F31DK10236101到KS）和美国心脏协会（CF和AG）的支持。

所有动物方案和实验过程由医学迈阿密大学米勒医学院大学的机构动物护理和使用委员会（IACUC）的批准。 1.准备<ol><li>预热肝灌注介质和肝脏，在37℃消化介质中的水浴中。 </li><li>设置一个灌注泵（30毫升/分钟）。仔细预填肝灌注介质泵的管道系统。避免在系统中的任何气泡，并准备立体显微镜。 </li><li>使用根据制造商的协议我型胶原涂层细胞培养皿。 </li></ol> 2.肝恢复<ol><li> 麻醉用氧气/异氟醚（异氟烷4％/ 2升/分钟氧气）供体小鼠。 <ol><li>通过降低异氟烷以2-2.5％或通过注射氯胺酮维持麻醉（100mg / kg的体重腹膜内）和赛拉嗪（10毫克/公斤体重的intraperitoneally）。连续异氟烷麻醉优选自氯胺酮降低血压，并可能导致肝毒性。 </li></ol></li><li>放置麻醉小鼠上的吸收垫。用胶带固定鼠标的四肢。 </li><li>刮脸和消毒鼠标的腹部，并从耻骨进行剖腹探查腹用手术剪刀锋利/钝头肝脏的颅边框。切开腹壁向两侧，膜片的尾部。 </li><li>经肠道仔细移动到右侧暴露下腔静脉（IVC）。用消毒棉签拭子和角度的尖镊子仔细解剖周围的IVC脂肪组织。 </li><li>切开肝上下隔膜，并用细手术剪刀锋利/锋利的尖端两侧切口打开胸腔。使用胸外科IVC丝（5/0）结扎。 </li><li>使用屏蔽静脉导管导管插入下腹IVC，小心地取出针头，连接灌注泵，开始灌注肝脏。如果执行得当，肝应立即开始发白和膨胀。 </li><li>横切门静脉使血液和灌注媒体适当的排水用细手术剪。 </li><li>灌注用约30毫升肝灌注介质的肝脏，然后切换到肝脏消化介质（30毫升）中。关闭灌注泵并取出插管灌注一次完成。 </li><li>轻轻暴露肝脏的中央区域并定位在连接组织联的肝叶。抓住中央连接光纤，仔细剖析控股肝脏全部到位组织和轻轻取出肝脏。 </li><li>紧接在肝脏转移到含有15毫升的50mL聚丙烯锥形管肝脏消化介质。 注:有没有具体的时间线的这一步。灌注后，肝脏保持在消化介质输送到无菌细胞培养罩。所有Subsequent步骤应在无菌环境中进行。 </li></ol>细胞3.隔离治疗<ol><li>在细胞培养罩，转移从50毫升聚丙烯锥形管含有消化媒体肝脏进入无菌组织培养皿。 </li><li>使用无菌镊子轻轻撕肝脏的四个叶片。如果正确执行，消化中应该由于从肝脏分离肝细胞变浑浊。 </li><li>用25毫升无菌枪头，通过70微米尼龙细胞过滤混浊消化介质过滤到一个新的50毫升聚丙烯锥形管以除去未消化的组织颗粒和细胞碎片。 </li><li>重复步骤3.2和3.3，用无菌磷酸盐缓冲盐水（PBS）。这有助于从肝驱除残存肝细胞。 </li><li>离心机在50×g离心在4℃下2分钟。吸出上清液含有过量的细胞碎片，轻轻地再暂停25毫升冷的1×细胞威廉姆斯的中E. </li><li>用25毫升无菌枪头，通过一个新的70微米尼龙细胞过滤器过滤再悬浮到50mL聚丙烯锥形管以实现清洁悬浮液。 </li><li>重复步骤3.5和3.6使用冷威廉姆斯中等E 1X，以获得更清晰的细胞悬液三个额外的洗涤。 </li><li>最后洗涤步骤后，吸去上清液，以除去过量的细胞碎片。重悬和过滤器的细胞在25mL暖Williams的媒体E 1X包含原代肝细胞的解冻和电镀补充剂通过一个新的70微米尼龙细胞过滤到50mL聚丙烯锥形管中。 </li><li>算使用血球25毫升细胞悬浮液中的细胞。平均预期15 - 每位成人肝20000000细胞。使用台盼蓝染色测定细胞生存力。 </li><li>板块细胞威廉姆斯的2毫升媒体E 1X包含原代肝细胞解冻和电镀添加剂在胶原包被的6孔策LL培养簇（每孔9×10 5个细胞）。 </li><li> 4小时后，介质改为威廉姆斯的媒体E 1X包含原代肝细胞的维护补充剂。 </li><li> 24小时后，血清饥饿用Dulbecco改进的Eagle培养基（DMEM）培养基1×6小时的细胞。 </li><li>用PBS处理细胞作为溶媒，FGF23（25毫微克/毫升），脂多糖（100微克/毫升），或在无血清培养基中的IL-6（50毫微克/毫升），并培育24小时。 </li></ol> 4.隔离的RNA <ol><li>使用根据制造商的说明在商业自旋柱试剂盒制备的RNA。 </li></ol> 5.生成的cDNA通过逆转录RNA分离<ol><li>添加200毫微克分离的RNA样品以PCR管。 </li><li>从步骤5.1添加核酸内切酶游离H 2 O至PCR管的14微升。 </li><li>从步骤5.1添加4微升反转录酶对PCR管。 </li><li>轻轻重悬内容，以混合均匀;离心平缓。 </LI&gt; <li>放置装有该混合物成热循环PCR管。用于逆转录运行热循环:1个循环的25℃（5分钟），42℃（30分钟），85℃（5分钟），然后保持在4℃。最终产品将是所需cDNA。 </li></ol> 6.实时定量PCR细胞分析（定量PCR） <ol><li> 准备96孔板。 <ol><li>放置在冰上的qPCR反应的所有成分:1μL的cDNA，0.25微米的正向引物，0.25μM反向引物，5μL的SYBR绿，3.5微升的PCR级水，以10μL的总体积。使用的SYBR Green，准备预混一式三份运行的所有样本。 </li><li>一旦主结构完成后，旋涡，使均匀。等分试样9的qPCR的微升主混合物到96-WLL板的各孔中。后，小心加cDNA的1微升到每口井。反应总量将成为10微升。 </li><li>加载完成后，密封用光学96孔平板粘合膜和短暂离心96孔板（50×g离心1分钟）。 </li></ol></li><li> 运行样品在实时PCR仪。处密封保存96孔板为实时PCR仪和运行样品按照仪器制造商的建议。标准和快速循环的例子包括在下面。 <ol><li>对于标准循环参数，使用以下命令:预变性94℃120秒，然后94℃40个循环15秒，60℃60秒，然后有选择地在4℃举行。 </li><li>对于快速循环参数:使用以下命令:94℃预变性30秒，然后95℃40个循环5秒，58℃15秒，然后72℃10秒。 </li></ol></li><li>参考仪器手册，了解有关如何分析数据的指导。 </li></ol> 7.通过ELISA细胞上清液的分析注:根据制造商的协议执行的所有步骤。 </p&gt; <ol><li> 样品制备 <ol><li>稀释分装398微升到单独的试管。吸取2μL细胞上清液样品放入398微升稀释剂管。这将提供为200的稀释倍数。 </li><li>对每个样品重复步骤7.1.1进行测试。 </li></ol></li><li> 程序 <ol><li>吸取100μL的200倍稀释的样品和标准于96孔板的孔中。 </li><li>孵育96孔板在25℃下用在以150rpm微孔板摇床上45分钟。 </li><li>洗孔5次用1×洗涤缓冲液。一旦最后一次洗涤完成后，取出用吸水纸全部残留洗涤液。 </li><li>加入酶标试剂500微升到每口井。 </li><li>重复步骤7.2.2 - 7.2.3。 </li><li>吸取100μL的四甲基联苯胺（TMB）试剂到96孔板的每个孔中。 </li><li>轻轻在25℃的微量混合96孔板20分钟摇床在150rpm。通过直接加入100微升的终止溶液以每孔停止反应，并轻轻混合。一旦停止溶液的颜色应该变黄。这个通知的终止溶液已正确混合。 </li><li>使用微量滴定板读数器，确定第一个5分钟内450nm处的光密度。 </li></ol></li><li> 结果计算 <ol><li>计算每个样本和每个标准平均吸光度值（A 450）的表。 </li><li>在NG密谋反对它的标准浓度每个标准的A 450平均组装的标准曲线/毫升的线性图。允许x轴表示的浓度和y轴来表示A 450值。 </li><li>利用意味着从每个稀释样品A 450，从标准曲线确定毫微克/毫升所需蛋白的浓度。 </li><li>通过稀释事实上乘以样品浓度河这将允许用于确定细胞上清液样品中的所希望的蛋白质的实际浓度。 </li><li>绘制从样本构建表成线图。如果OD 450值在标准曲线的外部，样品应适当地稀释并重新测试。 </li><li>结果归为500,000个细胞。 </li></ol></li></ol>

<ol>
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从小鼠中分离原代肝细胞是一种快速，廉价和可靠的工具来研究炎症反应体外。如果正确执行，结果可以很容易地生成并及时和具有成本效益的方式再现。以下几点应仔细评估，以确保成功隔离。 手术切口和下腔静脉的插管应在全身麻醉下，不安乐死之后进行。一个年轻的，缺乏经验的调查员将需要更多的时间在一开始就熟悉小鼠腹部的解剖学特征，并进行正确的插?...

慢性肾病（CKD）可以被定义为急性和慢性炎症1的状态。在慢性肾脏病患者中，phosphaturic激素成纤维细胞生长因子23（FGF23）的血清水平逐步上升，以维持血清磷酸盐稳态2。提高血清FGF23水平分别与心血管发病率和死亡率谁开始血液透析治疗3，4患者有关。此外，一些临床研究表明升高的FGF23水平与C-反应蛋白（CRP），白介素-6（IL-6）和肿瘤坏死因子α（TNFα）5,6的血清水平之间存在很强的相关性。另外，在实验研究中，我们最近表明，FGF23可以直接靶向肝细胞，并导致增加的CRP和IL-6 P的炎症反应roduction在肝脏7。因此，FGF23可能充当有助于全身炎症反应在CKD循环因素。 在70年代初，原代肝细胞分离并研究首次8。自那时以来，原代培养的肝细胞已被广泛用来研究代谢加工，激素功能，药物代谢和毒性，以及免疫和炎症反应9,10。先前协议已经主要描述原代肝细胞的酶活隔离从人肝脏组织11，12。虽然一个很好的模式，这留下了研究操纵基因是如何影响肝脏复杂的信号机制，以及在不同类型的刺激功能的后果的能力。在下面，我们描述了小鼠原代肝细胞的分离。值得注意的是，肝急性期反应的几种介质，如脂多糖（LPS），IL-6和FGF23的效果可在简单，快速和可重复的方式13进行分析。 在此，我们提出了从成年小鼠肝细胞的酶促隔离的协议，并且我们表明，炎症的建立诱导，如LPS和IL-6，以及新颖的炎症介质如FGF23，可直接刺激的表达和分泌炎性细胞因子，如在培养的肝细胞的CRP和IL-6。

<table border="0" cellpadding="0" cellspacing="0" width="924">
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			<td height="21" style="height:21px;width:363px;">Consumables</td>
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			<td height="21" style="height:21px;">Cell Strainer 70 &#956;m Nylon cell strainer</td>
			<td>Falcon</td>
			<td>352350</td>
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			<td height="21" style="height:21px;">BD Insyte Autoguard</td>
			<td>BD</td>
			<td>381412</td>
			<td></td>
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			<td height="21" style="height:21px;">50 mL Polypropylene Conical Tube</td>
			<td>Falcon</td>
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			<td height="21" style="height:21px;">1cc U-100 Insulin Syringe 28 G 1/2</td>
			<td>Becton Dickinson</td>
			<td>329420</td>
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			<td height="21" style="height:21px;">Tissue Culture Dish 100 x 20 mm Style</td>
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			<td>353003</td>
			<td></td>
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			<td height="21" style="height:21px;">6 well Cell Culture Cluster</td>
			<td>Costar</td>
			<td>3516</td>
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			<td height="21" style="height:21px;">5/0 Black Braided Surgical Silk (100 yards)</td>
			<td>LOOK</td>
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			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
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			<td height="21" style="height:21px;">Equipment</td>
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			<td>Gilson</td>
			<td>F155007</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Hemacytometer Kits, Propper</td>
			<td>VWR</td>
			<td>48300-474</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Hemacytometer Cover Glasses, Propper</td>
			<td>VWR</td>
			<td>48300-470</td>
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			<td height="21" style="height:21px;">Surgical Scissers - Sharp/Blunt</td>
			<td>F.S.T.</td>
			<td>14001-12</td>
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			<td>F.S.T.</td>
			<td>14058-11</td>
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			<td height="22" style="height:23px;">Dumont SS-45 Forceps</td>
			<td>F.S.T.</td>
			<td>11203-25</td>
			<td></td>
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			<td height="22" style="height:23px;">Student Tissue Forceps</td>
			<td>F.S.T.</td>
			<td>991121-12</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reagents</td>
			<td></td>
			<td></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Acetic acid solution, 2.0 N</td>
			<td>Sigma</td>
			<td>&#160;&#160;&#160; A8976-100ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoflurane, USP 250 mL</td>
			<td>Piramal Healthcare</td>
			<td>&#160;&#160; 66794-013-25</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KetaVed 1000mg/10mL (100mg/mL)</td>
			<td>VEDCO</td>
			<td>&#160;&#160;&#160; 50989-161-06</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Xylazine 100 mg/mL</td>
			<td>AnaSed Injection</td>
			<td>139-236</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Willams&#39; Medium E (1x)</td>
			<td>gibco</td>
			<td>12551-032</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Liver Perfusion Medium (1x)</td>
			<td>gibco</td>
			<td>17701-038</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Liver Digest Medium (1x)</td>
			<td>Life Technologies</td>
			<td>17703034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Primary Hepatocyte Thawing and Plating Supplements</td>
			<td>Life Technologies</td>
			<td>CM3000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Primary Hepatocyte Maintenance Supplements</td>
			<td>Life Technologies</td>
			<td>CM4000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffer Saline (PBS) pH 7.4</td>
			<td>ThermoFisher scientific</td>
			<td>10010031</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagen Type 1</td>
			<td>Corning</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypan Blue Solution</td>
			<td>VWR</td>
			<td>45000-717</td>
			<td></td>
		</tr>
	</tbody>
</table>

induction of an inflammatory response in primary hepatocyte cultures from mice

肝脏在全身性炎症的调节决定性的作用。特别是在慢性肾脏疾病，肝脏响应于尿毒症环境，氧化应激，内毒素血症和通过产生大量的急性期反应物的循环促炎性细胞因子的清除率降低反应。实验工具来研究炎症和肝细胞的基本作用是理解的调节和肝细胞因子的贡献全身急性期反应和延长的促炎症的情况下，尤其是在复杂的环境至关重要如慢性肾脏疾病。自体内研究炎症的复杂的机制仍然是具有挑战性的，耗费资源的，通常需要转基因动物的使用，一次分离的肝细胞提供一个强大的工具来获得机械分析上市肝急性期反应。由于这种体外技术特点适中成本，高重可生产和技术常识，一次分离的肝细胞也可以很容易地用作筛选方法。这里，我们描述一个基于酶促法以分离原代鼠肝细胞，和我们描述在使用ELISA和定量实时PCR这些细胞的炎症反应的评估。

组织学一次分离和培养的细胞的代表性光显微镜图像在图1A中描绘。免疫细胞化学分析表明，分离的肝细胞中高度表达白蛋白（红色），以及成纤维细胞生长因子受体-4（FGFR4）（绿色）。细胞核用4&#39;，6-二脒基-2-苯基吲哚（DAPI）（蓝色）。 （ 图1B）。 定量?...

Watch this Scientific Journal Video about Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice at JoVE.com

Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

这里，我们表明酶促方法来原代肝细胞从成年小鼠隔离，我们描述了使用ELISA和实时PCR的炎症反应的量化。

从小鼠在原代肝细胞培养物的炎性反应的诱导

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the ...

induction-an-inflammatory-response-primary-hepatocyte-cultures-from

Research

JoVE Journal

Immunology and Infection

9.9K 次观看.  University of Miami Leonard M. Miller School of Medicine. 这里，我们表明酶促方法来原代肝细胞从成年小鼠隔离，我们描述了使用ELISA和实时PCR的炎症反应的量化。 

视频: Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse models allow us to study how diseases unfold, often providing a good replica of the same processes occurring in humans. For some autoimmune diseases, like type 1A diabetes, there are models (the NOD mouse) that spontaneously develop a disease similar to the human counterpart. For many other autoimmune diseases, however, the model needs to be induced experimentally. A common approach in this regard is to inject the mouse with a dominant antigen derived from the organ being studied. For example, investigators interested in autoimmune thyroiditis inject mice with thyroglobulin2, and those interested in myasthenia gravis inject them with the acetylcholine receptor3. If the autoantigen for a particular autoimmune disease is not known, investigators inject a crude protein extract from the organ targeted by the autoimmune reaction. For autoimmune hypophysitis, the pathogenic autoantigen(s) remain to be identified4, and thus a crude pituitary protein preparation is used. In this video article we demonstrate how to induce experimental autoimmune hypophysitis in SJL mice.

This video shows how to induce autoimmune hypophysitis in SJL mice and how to assess its severity by histopathology.

SJL小鼠诱导实验性自身免疫性垂体炎

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse ...

科研

免疫与感染

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

一种简单而有效的隔离混合原代培养成人肝细胞巨噬细胞的方法

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates epithelial differentiation. There are many uses for this system. For instance, the ability to grow three-dimensional organotypic raft cultures of keratinocytes has been an important milestone in the study of human papillomavirus (HPV)1. The life cycle of HPV is tightly linked to the differentiation of squamous epithelium2. Organotypic epithelial raft cultures as demonstrated here reproduce the entire papillomavirus life cycle, including virus production3,4,5. In addition, these raft cultures exhibit dysplastic lesions similar to those observed upon in vivo infection with HPV. Hence this system can also be used to study epithelial cell cancers, as well as the effect of drugs on epithelial cell differentiation in general. Originally developed by Asselineau and Prunieras6 and modified by Kopan et al.7, the organotypic epithelial raft culture system has matured into a general, relatively easy culture model, which involves the growth of cells on collagen plugs maintained at an air-liquid interface (Figure 1A). Over the course of 10-14 days, the cells stratify and differentiate, forming a full thickness epithelium that produces differentiation-specific cytokeratins. Harvested rafts can be examined histologically, as well as by standard molecular and biochemical techniques. In this article, we describe a method for the generation of raft cultures from primary human keratinocytes. The same technique can be used with established epithelial cell lines, and can easily be adapted for use with epithelial tissue from normal or diseased biopsies8. Many viruses target either the cutaneous or mucosal epithelium as part of their replicative life cycle. Over the past several years, the feasibility of using organotypic raft cultures as a method of studying virus-host cell interactions has been shown for several herpesviruses, as well as adenoviruses, parvoviruses, and poxviruses9. Organotypic raft cultures can thus be adapted to examine viral pathogenesis, and are the only means to test novel antiviral agents for those viruses that are not cultivable in permanent cell lines.

An in vitro method to mimic in vivo epithelial differentiation is described. Many viruses target epithelial cells as part of their viral life cycle, and this method provides a means of examining virus:host interactions that more closely resembles that which occurs in vivo. This technique can be used with primary keratinocytes, established cell lines, as well as normal or diseased biopsy tissue.

从小学的人角质形成细胞器官筏文化的代

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates ...

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity 1,2. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection 3. Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop 4-6. As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted.
	Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study.
	The principle and protocol of this methodology have been described in the literature 7,8. Additionally, alternate methodologies to isolate primary microglia are well described 9-12. Homogenized brain tissue may be separated by density gradient centrifugation to yield primary microglia 12. However, the centrifugation is of moderate length (45 min) and may cause cellular damage and activation, as well as, cause enriched microglia and other cellular populations. Another protocol has been utilized to isolate primary microglia in a variety of organisms by prolonged (16 hr) shaking while in culture 9-11. After shaking, the media supernatant is centrifuged to isolate microglia. This longer two-step isolation method may also perturb microglial function and activation. We chiefly utilize the following microglia isolation protocol in our laboratory for a number of reasons: (1) primary microglia simulate in vivo biology more faithfully than immortalized rodent microglia cell lines, (2) nominal mechanical disruption minimizes potential cellular dysfunction or activation, and (3) sufficient yield can be obtained without passage of the mixed glial cell cultures.
	It is important to note that this protocol uses brain tissue from neonatal rat pups to isolate microglia and that using older rats to isolate microglia can significantly impact the yield, activation status, and functional properties of isolated microglia. There is evidence that aging is linked with microglia dysfunction, increased neuroinflammation and neurodegenerative pathologies, so previous studies have used ex vivo adult microglia to better understand the role of microglia in neurodegenerative diseases where aging is important parameter. However, ex vivo microglia cannot be kept in culture for prolonged periods of time. Therefore, while this protocol extends the life of primary microglia in culture, it should be noted that the microglia behave differently from adult microglia and in vitro studies should be carefully considered when translated to an in vivo setting.

Isolating primary microglia from the cellular heterogeneity of the brain is essential to investigate their role in both physiological and pathological conditions. This protocol describes a mechanical isolation and mixed cell culture technique that provides high yield and high purity, viable primary microglial cells for in vitro study and downstream applications.

初级小胶质细胞的分离，从新生大鼠脑组织胶质细胞混合文化

Microglia account for approximately 12% of the  total cellular population in the mammalian brain. While neurons and astrocytes are considered  the major ...

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

区分功能基因表达的作用，通过骨髓移植免疫和非免疫细胞在小鼠结肠炎

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of human Type 1 diabetes (T1D). Cell transfer studies in irradiated recipient mice have established that T cells are pivotal in T1D pathogenesis in this model. We describe herein a simple method to rapidly induce T1D by adoptive transfer of purified, primary CD4+ T cells from pre-diabetic NOD mice transgenic for the islet-specific T cell receptor (TCR) BDC2.5 into NOD.SCID recipient mice. The major advantages of this technique are that isolation and adoptive transfer of diabetogenic T cells can be completed within the same day, irradiation of the recipients is not required, and a high incidence of T1D is elicited within 2 weeks after T cell transfer. Thus, studies of pathogenesis and therapeutic interventions in T1D can proceed at a faster rate than with methods that rely on heterogenous T cell populations or clones derived from diabetic NOD mice.

We provide a reproducible method to induce type 1 diabetes (T1D) in mice within two weeks by the adoptive transfer of islet antigen-specific, primary CD4+ T cells.

加速1型糖尿病感应在小鼠体内的过继转移致糖尿病的CD4 + T细胞

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

隔离和小鼠骨髓的静脉注射单核细胞衍生

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

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 ...

Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory response by the endothelial layer is crucial to efficiently fight against harmful inputs and aid in the recovery of damaged areas. When the endothelial cells are exposed to an inflammatory environment, such as the outer component of gram-negative bacteria membrane, lipopolysaccharide (LPS), they express soluble pro-inflammatory cytokines, such as Ccl5, Cxcl1 and Cxcl10, and trigger the activation of circulating leukocytes. In addition, the expression of adhesion molecules E-selectin, VCAM-1 and ICAM-1 on the endothelial surface enables the interaction and adhesion of the activated leukocytes to the endothelial layer, and eventually the extravasation towards the inflamed tissue. In this scenario, the endothelial function must be tightly regulated because excessive or defective activation in the leukocyte recruitment could lead to inflammatory-related disorders. Since many of these disorders do not have an effective treatment, novel strategies with a focus on the vascular layer must be investigated. We propose comprehensive assays that are useful to the search of novel endothelial regulators that modify leukocyte function. We analyze endothelial activation by using specific expression targets involved in leukocyte recruitment (such as, cytokines, chemokines, and adhesion molecules) with several techniques, including: real-time quantitative polymerase chain reaction (RT-qPCR), western-blot, flow cytometry and adhesion assays. These approaches determine endothelial function in the inflammatory context and are very useful to perform screening assays to characterize novel endothelial inflammatory regulators that are potentially valuable for designing new therapeutic strategies.

Vascular endothelium tightly controls leukocyte recruitment. Inadequate leukocyte extravasation contributes to human inflammatory diseases. Therefore, searching for novel regulatory elements of endothelial activation is necessary to design improved therapies for inflammatory disorders. Here, we describe a comprehensive methodology to characterize novel endothelial regulators that can modify leukocyte trafficking during inflammation.

用于炎症反应的新内皮调节因子的筛选测定

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory ...

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human central nervous system (CNS) autoimmune demyelinating disease, creation of an AQP4-targeted model with both clinical and histologic manifestations of CNS autoimmunity has proven challenging. Immunization of wild-type (WT) mice with AQP4 peptides elicited T cell proliferation, although those T cells could not transfer disease to na&#239;ve recipient mice. Recently, two novel AQP4 T cell epitopes, peptide (p) 135-153 and p201-220, were identified when studying immune responses to AQP4 in AQP4-deficient (AQP4-/-) mice, suggesting T cell reactivity to these epitopes is normally controlled by thymic negative selection. AQP4-/- Th17 polarized T cells primed to either p135-153 or p201-220 induced paralysis in recipient WT mice, that was associated with predominantly leptomeningeal inflammation of the spinal cord and optic nerves. Inflammation surrounding optic nerves and involvement of the inner retinal layers (IRL) were manifested by changes in serial optical coherence tomography (OCT). Here, we illustrate the approaches used to create this new in vivo model of AQP4-targeted CNS autoimmunity (ATCA), which can now be employed to study mechanisms that permit development of pathogenic AQP4-specific T cells and how they may cooperate with B cells in NMO pathogenesis.

Here, we present a protocol to induce paralysis and opticospinal inflammation by transfer of aquaporin-4 (AQP4)-specific T cells from AQP4-/- mice into WT mice. In addition, we demonstrate how to use serial optical coherence tomography to monitor visual system dysfunction.

诱导麻痹及视觉系统损伤小鼠 T 细胞特异的视神经脊髓炎抗原水通道蛋白-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...