这项工作是由国家过敏与感染疾病格兰特R01AI079253（以BHS）的支持，以及由美国国家癌症研究所癌症中心支援津贴CA016056到罗斯威尔公园癌症研究所。

动物在这项研究中执行的所有程序批准了动物护理和使用委员会在罗斯威尔公园癌症研究所和遵守所有州，联邦和卫生法规国家机构。 1， 在体内巨噬细胞标记注意：PHK26是将要采取由吞噬细胞在血液循环和在组织中静脉内给药（IV）时，亲脂性染料。 PKH26用于体内标记，以减少收获后处理的巨噬细胞的数量。 PKH-26具有稳定地积聚在肺泡巨噬细胞的延长期的优势。给药后10天等待允许从循环只留下肺泡巨噬细胞稳定标记PKH26 8,9所有标记细胞的间隙。 PKH26是一个红色的荧光，具有激发（551 nm）和发射（567 nm）的特性兼容罗丹明或藻红蛋白的检测系统。任何标记过程可以介绍的神器，其中包括生存能力的丧失。然而，由于我们使用的巨噬细胞的标记以同一方法，不同的鼠标基因型（ 例如 ，WT与NADPH氧化酶缺陷），这些文物的影响将是统一的。 <ol><li> PKH26稀释的原液（乙醇1×10 -3 m）在由制造商提供的稀释液1:5。 </li><li>负载100μl的稀释PKH26成1/2 20ng/ml胰岛素注射器（29克X 1/2“）对每只小鼠。 </li><li>通过静脉注射（或尾静脉或眶）至少10天，收获前管理到小鼠。 </li></ol> 2，收获肺泡巨噬细胞支气管肺泡灌洗（BAL） 小鼠用于实验的数目可以变化。一个典型的产量从未受刺激的小鼠是在3-5×10 5肺泡巨噬细胞的范围内，但这个数字也相差很大BAS版上的因素，如进行灌洗者的鼠标，经验大小和灌洗液和从肺中排出的体积的滴注的数量。在这个协议中肺灌洗是一个封闭胸腔它伴随着反复1毫升灌洗有助于提高细胞产量进行。 <ol><li>通过使用无菌条件下，保持溶液的无菌管理致死剂量阿佛丁麻醉剂（12.5毫克），腹腔（IP）安乐死鼠标。 </li><li>用70％乙醇彻底喷鼠标。 </li><li>从动物的颅年底去除皮肤暴露胸部（ 图1A）。 <ol><li>做一个小切口在皮肤只是隔膜下方。 </li><li>将手指插入切口，拉皮肤远离动物（ 图1A）的颅结束。 </li><li>向上推略微的头，延长颈部腹侧（ 图1A）。 </li></ol></li><li>通过切左肋一个小孔进入右侧胸廓放气肺和也允许在以下步骤中的肺的可视化。 </li><li>找到气管，并认真剖析了周围的组织。 <ol><li>取出唾液腺，胸骨舌骨肌和喉等用弯钳。 </li><li>小心地抬起了弯钳和切去，用剪刀薄组织覆盖气管（外膜）揭示的基本环状软骨结构。 </li></ol></li><li>将导管插入气管。 <ol><li>用弯钳，放置缝合后面（背侧）的气管，并通过开口拉一个10cm的长度。 </li><li>启动环状（软骨环增厚）上面仔细地引导22政导管下来气管停在那里气管消失胸骨后面。 </li><li>紧紧绑周围缝合气​​管导管到舒适的位置。从导管取出针头。 </li></ol></li><li>装配4路阀。 <ol><li>填1个6毫升注射器用DPBS，1％FBS和附加到4路阀， 如图1B所示。 </li><li>附上一个空6毫升在注射器上的4路阀（ 图1B）所指示的位置。 </li><li>重视开放端口上的活塞，以导管4路（ 图1B）。注意不要从气管导管打跑。 </li></ol></li><li>从收集肺灌洗液。 注：调查人员可以选择使用肺泡灌洗液中下三卷，以尽量减少组织损伤的可能性。那同样的方法在所有小鼠组被贯彻应用是很重要的。 <ol><li>所以转空注射器被关闭的水龙头把手， 慢慢地注入约1mL DPBS，1％FBS膨胀的肺。通过切孔在步骤2.4观察肺胀。 </li><li>要小心，不要过度膨胀的肺。 </li><li>打开水龙头手柄，使之充分注射器被关闭，并仔细撤回流感<html> ID从肺部。 </li><li>观察肺放气通孔切割步骤1.4。导管可能需要移动稍微向后或向前来获得一个很好的借鉴。采取特殊的预防措施，以保持导管气管内。 </li><li>重复步骤2.8.1-2.8.4 5-6倍，直到所有的DPBS，1％FBS的已注入和从肺部排出。注意：肺液的回收将不注射液的100％。 </li></ol></li><li>空注射器灌洗液到50ml锥形管中。保持细胞在室温下。 </li><li>通过在300 XG离心5分钟，在室温下沉淀细胞，并倒出上清液。这样如果需要，可以保存用于细胞因子分析。 </li><li>重悬的细胞在1-5毫升的RPMI 1640含10％FBS和血细胞计数器计数。在未刺激的小鼠，&gt; 95％的细胞由BAL收获的预计是巨噬细胞，这可以通过细​​胞学进行确认。 </li></ol> 3，培养和收获真菌“&gt;在这个协议中使用的烟曲霉表达绿色荧光蛋白在其整个生命周期的各个阶段：孢子，germlings和菌丝这种真菌菌株是由马戈·摩尔博士，西门菲沙大学，伯纳比，不列颠哥伦比亚省，加拿大提供。 <ol><li>在沙氏脑心脏浸液（BHI）琼脂斜面用氯霉素（0.05克/升）和庆大霉素（0.5克/升）表达GFP的烟曲霉菌株的分生孢子盘。 </li><li>在孵育在室温下松瓶盖为7〜10天的黑暗。 </li><li>收获分生孢子通过倾斜洗涤用10毫升0.01％吐温20的DPBS。轻轻刮除木耳与stripette松动。冲洗斜几次，以增加产量。 </li><li>通过100微米的细胞过滤网通过分生孢子悬浮液置于50ml锥形。 </li><li>粒料分生孢子在400×g离心离心10分钟。 </li><li>分生孢子重悬在50毫升的DPBS和计数使用血球。 </li><li>用DPBS洗两次并稀释至所需浓度滤过。 </li></ol> 4，真菌挑战和共聚焦显微镜为了研究巨噬细胞NADPH氧化酶在宿主防御中的作用，从三种基因型控制吞噬曲霉菌孢子的生长小鼠分离的肺泡巨噬细胞的能力进行了评估。在本研究中使用的基因型包括; 1）野生型小鼠，2）NCF1 * / *锰小鼠的天然存在的突变NCF1（NCF1编码为P47 PHOX，吞噬细胞的NADPH氧化酶的必需组分）离开他们的NADPH氧化酶缺陷型，和3）NCF1 * / *锰+转基因小鼠（MN表示该转基因携带野生型NCF1人类CD68启动子的控制下），其中NADPH氧化酶活性已被重新溶解在单核细胞/巨噬细胞群10，11。 共聚焦显微镜最适合于该试验中，由于存在和在幻灯片非吞噬的分生孢子的生长。共焦显微镜将允许在Z轴中的各个平面的可视化，并且使真菌生长围绕，而不是内巨噬细胞之间的差异。所有的共聚焦图像是使用63X油浸镜头获得的。 Z-栈收购与2.5电子的变焦倍数。一个Z-堆栈的厚度是用DAPI和亮场图像的定位字段的顶部和底部的确定。 Z-堆栈介乎14至24微米，个别切片〜1微米厚。巨噬细胞的定量，1X电子变焦的单扫描每基因型的10个字段分别进行。被确定完整的巨噬细胞的基础上分别PKH26和膜和细胞核的DAPI染色。分生孢子的基础上确定的FITC表达和亮场图像。 <ol><li>准备无菌盖玻片和板<ol><li>下一个生物安全柜，浸泡22×22毫米的玻璃盖玻片在70％桥亚乙基升5-10分钟。 </li><li>在无菌PBS冲洗盖玻片。 </li><li>用无菌镊子，轻轻地放在一个单独的盖玻片在无菌单独包装的6孔板的每个孔的底部。 </li></ol></li><li>种子〜1×10 6个收获的PKH26标记的肺泡巨噬细胞悬浮于300微升的RPMI 1640，10％FBS到每个盖玻片。取决于收获产量的确切数量可以改变。 </li><li>让巨噬细胞在37℃温育，用5％的CO 2过夜以粘附。 </li><li>第二天早晨，加〜1×10 7表达GFP的A。曲霉分生孢子悬浮于100μl预热的（37℃）的RPMI 1640，10％FBS。 </li><li>返回板到37℃培养箱中，用5％的CO 2。 </li><li>细胞应该被固定为至少2小时，室温过夜或在4℃下在3,7和14小时修复细胞，加入2％的甲醛等体积到各孔中（最终浓度1％的甲醛）。地方板的在4℃下早期时间点过夜。在最后的时间点（14小时），固定的细胞在室温下搅拌2小时。 </li><li>固定后，使用镊子轻轻取出盖玻片和冲洗的DPBS。 </li><li>放置盖玻片的边缘折叠的Kimwipe去除多余的液体。 </li><li>适用于6微升荧光封固剂用DAPI到玻璃显微镜载片。 </li><li>莱盖玻片的一个边缘上的幻灯片，并与细胞侧轻轻下拉到安装介质。安装介质将蔓延并形成均匀的一层盖玻片下方。有没有必要压在盖玻片。 </li><li>密封盖玻片的边缘与透明指甲油，并在空气中晾干。 </li><li>店准备的幻灯片在幻灯片框（避光），在室温下，直到准备通过共聚焦显微镜来分析。 </li></ol>

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作者什么都没有透露。

使用这种方法进行体外分析巨噬细胞的抗真菌活性连同体内真菌的挑战，我们先前表明，在巨噬细胞NADPH氧化酶起着抵御A的重要作用烟4。使用隔离的肺泡巨噬细胞使我们能够专注于他们的抗真菌活性在没有其他免疫细胞（ 如中性粒细胞招募）， 在体内防御曲霉 。 各种可视化策略已经被用于评价巨噬细胞的抗真菌活性<e...

肺泡巨噬细胞是遇到吸入微生物一线吞噬细胞。巨噬细胞识别黑图案由病原体识别受体，摄取并限制吸入孢子（分生孢子）的生长，并启动炎症反应。吞噬细胞NADPH氧化酶转化分子氧超氧阴离子和下游活性氧中间体（投资回报）​​。慢性肉芽肿病（CGD）是NADPH氧化酶的特征是严重的细菌和真菌感染和由过度的炎症反应的一种遗传性疾病。 虽然NADPH氧化酶是至关重要的中性粒细胞介导的宿主防御1 - 3，NADPH氧化酶的巨噬细胞中的重要性是不明确的。此前的研究表明，肺泡巨噬细胞摄取并杀死曲霉菌孢子，而中性粒细胞主要针对菌丝阶段5。但是，也出现了相互矛盾的雷苏尔TS作为NADPH氧化酶在巨噬细胞中控制A的生长的作用曲霉孢子6,7。 该方法的目标是界定在介导针对A.宿主防御NADPH氧化酶的巨噬细胞中的具体作用烟曲霉菌孢子。我们发现，在肺泡巨噬细胞NADPH氧化酶控制吞噬A的增长烟曲霉菌孢子4。为了最准确地模拟主机对吸入真菌，我们使用了支气管肺泡灌洗收集肺泡巨噬细胞的未刺激小鼠紧随牺牲。使用隔离的肺泡巨噬细胞使我们能够专注于自己的抗真菌活性在没有其他免疫细胞（ 如中性粒细胞招募）， 在体内防御曲霉 。在该方法中，肺泡巨噬细胞进行染色在事先体内收获，以尽量减少收获后的操作量。&lt;/ P&gt; 另一个优点这个协议是使用GFP表达A的曲霉菌株。这种真菌表达从分生孢子阶段GFP通过菌丝阶段，并没有必要进一步染色。以获得具有更详细的图像，我们选择了共焦显微镜使从所获得的图像的三维结构的重建。三维重建给出菌丝生长周围，而不是在或通过巨噬细胞之间进行区分的能力。分生孢子也可以精确地辨别作为细胞内与细胞外。

<table border="0" cellpadding="0" cellspacing="0" width="573">
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	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">PKH26GL-1KT</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2,2,2 Tribromoethanol</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">T48402</td>
			<td>Used to make AvertinAnesthetic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2-methyl-2-butanol</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">A-1685</td>
			<td>Used to make AvertinAnesthetic</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Dulbecco&#39;s Phosphate Buffered Saline (without calcium and magnesium)</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">21-031-CV</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">NH4CL</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">A0171</td>
			<td>Used to make ACK red cell lysis buffer</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">KHCO3</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">P91444</td>
			<td>Used to make ACK red cell lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EDTA</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">E6758</td>
			<td>Used to make ACK red cell lysis buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">22 gauge X 1.00 inch i.v. catheter</td>
			<td style="width:71px;">BD&#160;</td>
			<td align="right" style="width:119px;">381523</td>
			<td>Insyte Autoguard Winged</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">insulin syringes</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">6 ml syringes</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">4-way stopcock</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">50-700-077</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">suture thread</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50 ml conical centrifuge tubes</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">gauze sponges (4 inch square)</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RPMI 1640 with L-glutamine</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">10-040-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal Bovine Serum</td>
			<td style="width:71px;">Hyclone</td>
			<td style="width:119px;">SH30396.03</td>
			<td>heat inactivated</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hema 3 Stain Set</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">22-122-911</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cytoseal 60</td>
			<td style="width:71px;">Thermo Scientific</td>
			<td style="width:119px;">8310-4</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Sabouraud Brain Heart Infusion Agar with Chloramphenicol and Gentamicin Slants</td>
			<td style="width:71px;">BD&#160;</td>
			<td align="right" style="width:119px;">297252</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Aspergillus fumigatous strain expressing green fluorescent protein</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>provided by Dr Margo Moore, Simon Fraser University, Burnaby, BC, Canada</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">100 micron Cell Strainers</td>
			<td style="width:71px;">BD Falcon</td>
			<td style="width:119px;">64753-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">scissors</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">forceps</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Biological Safety Cabinet Class II</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sorval ST40 Centrifuge with swing bucket rotor</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Leica DM1000 light microscope</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hemocytometer</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cytospin 2 centrifuge</td>
			<td style="width:71px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Cell Culture Incubator</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>37 &#176;C, 5%&#160; CO2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">22X22 mm micro cover glass</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">48366-227</td>
			<td>glass coverslips</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">6 well Cell Culture Plates</td>
			<td style="width:71px;">Corning</td>
			<td align="right" style="width:119px;">3506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10% Neutral Buffered Formalin</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">BDH0502-1LP</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Vectashield Mounting medium with DAPI</td>
			<td style="width:71px;">Vector Laboratories</td>
			<td style="width:119px;">H-1200</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Leica TCS SP2 system with a laser point scanner</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>Mounted on DMIRE2 fluorescence microscope</td>
		</tr>
	</tbody>
</table>

assessing anti-fungal activity of isolated alveolar macrophages by confocal microscopy

肺是其中的宿主细胞经常接触到的微生物和微生物产品的接口。肺泡巨噬细胞是遇到吸入真菌和其它微生物的一线吞噬细胞。巨噬细胞和其他免疫细胞识别曲霉图案由病原体识别受体，并启动下游的炎症反应。吞噬细胞NADPH氧化酶产生活性氧中间体（投资回报）​​，是宿主防御的关键。虽然NADPH氧化酶是至关重要的中性粒细胞介导的宿主防御1 - 3，NADPH氧化酶的巨噬细胞中的重要性是不明确的。本研究的目标是界定在介导针对A.宿主防御NADPH氧化酶的巨噬细胞中的具体作用曲霉 。我们发现，在肺泡巨噬细胞NADPH氧化酶控制吞噬A的增长烟曲霉菌孢子4。在这里，我们描述了一种用于评估老鼠的能力lveolar巨噬细胞（AMS）来控制细胞吞噬黑曲霉的孢子（分生孢子）的生长。肺泡巨噬细胞进行染色体内并通过支气管肺泡灌洗（BAL）从小鼠十天后分离。巨噬细胞被镀到玻璃盖玻片上，然后种入绿色荧光蛋白（GFP）表达A.烟曲霉菌孢子。在指定的时间，将细胞固定和完好的巨噬细胞与吞噬孢子的数量通过共聚焦显微镜进行评估。

<html>从未经刺激的小鼠收集肺泡巨噬细胞通过BAL将导致基于细胞学一个纯度&gt; 95％的人口​​。在3和7小时（ 图2）的时间点之前的真菌生长的菌丝阶段，并允许对分生孢子的基因型之间的吞噬效率的比较。其中基因型可以是关于抑制吞噬的分生孢子于组织侵入菌丝阶段的过渡（ 图3）的能力进行比较，在14小时的时间点是。完好的巨噬细胞可以通过共聚焦显微镜的基础上...

Watch this Scientific Journal Video about Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy at JoVE.com

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

一种方法来评估分离的小鼠肺泡巨噬细胞，激光共聚焦显微镜，以控制吞噬曲霉菌孢子的生长的能力。

评估隔离肺泡巨噬细胞的激光共聚焦显微镜抗真菌活性

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

assessing-anti-fungal-activity-isolated-alveolar-macrophages-confocal

Research

JoVE Journal

Immunology and Infection

11.4K 次观看.  Roswell Park Cancer Institute. 一种方法来评估分离的小鼠肺泡巨噬细胞，激光共聚焦显微镜，以控制吞噬曲霉菌孢子的生长的能力。 

视频: Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. Thus, in vivo imaging of malaria parasites during pre-erythrocytic stages have revealed the active entrance of parasites into skin lymph nodes 3, the complete development of the parasite in the skin 4, and the formation of a hepatocyte-derived merosome to assure migration and release of merozoites into the blood stream 5. Moreover, the development of individual parasites in erythrocytes has been recently documented using 4D imaging and challenged our current view on protein export in malaria 6. Thus, intravital imaging has radically changed our view on key events in Plasmodium development. Unfortunately, studies of the dynamic passage of malaria parasites through the spleen, a major lymphoid organ exquisitely adapted to clear infected red blood cells are lacking due to technical constraints.
Using the murine model of malaria Plasmodium yoelii in Balb/c mice, we have implemented intravital imaging of the spleen and reported a differential remodeling of it and adherence of parasitized red blood cells (pRBCs) to barrier cells of fibroblastic origin in the red pulp during infection with the non-lethal parasite line P.yoelii 17X as opposed to infections with the P.yoelii 17XL lethal parasite line 7. To reach these conclusions, a specific methodology using ImageJ free software was developed to enable characterization of the fast three-dimensional movement of single-pRBCs. Results obtained with this protocol allow determining velocity, directionality and residence time of parasites in the spleen, all parameters addressing adherence in vivo. In addition, we report the methodology for blood flow quantification using intravital microscopy and the use of different colouring agents to gain insight into the complex microcirculatory structure of the spleen. 
Ethics statement
All the animal studies were performed at the animal facilities of University of Barcelona in accordance with guidelines and protocols approved by the Ethics Committee for Animal Experimentation of the University of Barcelona CEEA-UB (Protocol No DMAH: 5429). Female Balb/c mice of 6-8 weeks of age were obtained from Charles River Laboratories.

We show the method for performing intravital microscopy of the spleen using GFP transgenic malaria parasites and the quantification of parasite mobility and blood flow within this organ.

活体显微镜的脾：寄生虫的流动性和血流量的定量分析

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

科研

免疫与感染

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

在髓系细胞动态实时成像<em&gt;装甲运兵车<sup&gt;最小/ +</sup</em&gt;肠道肿瘤通过旋转盘共聚焦显微镜

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

人THP-1巨噬细胞通过核转高效转染

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

Proteomic Profiling of Macrophages by 2D Electrophoresis

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The key role of macrophages has been shown in various pathological disorders such as inflammatory, immunological, and infectious diseases. In this protocol, we use primary cultures of human monocyte-derived macrophages that can be differentiated into the M1 (pro-inflammatory) or the M2 (anti-inflammatory) phenotype. This in vitro model is reliable for studying the biological activities of M1 and M2 macrophages and also for a proteomic approach. Proteomic techniques are useful for comparing the phenotype and behaviour of M1 and M2 macrophages during host pathogenicity. 2D gel electrophoresis is a powerful proteomic technique for mapping large numbers of proteins or polypeptides simultaneously. We describe the protocol of 2D electrophoresis using fluorescent dyes, named 2D Differential Gel Electrophoresis (DIGE). The M1 and M2 macrophages proteins are labelled with cyanine dyes before separation by isoelectric focusing, according to their isoelectric point in the first dimension, and their molecular mass, in the second dimension. Separated protein or polypeptidic spots are then used to detect differences in protein or polypeptide expression levels. The proteomic approaches described here allows the investigation of the macrophage protein changes associated with various disorders like host pathogenicity or microbial toxins.

Macrophages are the key cells involved in host pathogenicity. Macrophages display phenotypic and functional diversity that can be analysed and detected by proteomic analysis. This article describes how to perform 2D electrophoresis of primary cultures of human macrophages differentiated into M1 or M2 phenotype.

巨噬细胞通过二维电泳蛋白组学分析

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

代和GM-CSF的鉴定源性肺泡状巨噬细胞和树突状细胞从小鼠骨髓

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

This manuscript describes a detailed protocol for phenotypic and quantitative analysis of resident macrophages from rat kidneys by flow cytometry. The resulting stained cells can be also used for other applications, including cell sorting, gene expression analysis or functional studies, thus increasing the information obtained in the experimental model.

通过流式细胞仪大鼠肾脏巨噬细胞的表型特征

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.

受肥胖相关炎症组织的巨噬细胞的分离、鉴定和纯化

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal microscopy methods to visualize macrophage extracellular traps (METs). These extracellular traps are defined by the presence of extracellular chromatin with co-expression of other components such as granule proteases, citrullinated histones, and peptidyl arginase deiminase (PAD). The expression of METs is generally measured after exposure to a stimulus and compared to un-stimulated samples. Samples are also included for background and isotype control. Cells are analyzed using well-defined image analysis software. Confocal microscopy may be used to define the presence of METs both in vitro and in vivo in lung tissue.

Macrophage extracellular traps are a newly described entity. This article will concentrate on confocal microscopy methods and how they are visualized in vitro and in vivo from lung samples.

利用共焦显微镜可视化巨噬细胞胞外陷阱

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

小鼠与人肺泡巨噬细胞的分离和体外培养

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of the actin cytoskeleton to capture, envelop and engulf large particles. Although phagocytic receptors, downstream signaling pathways, and effectors, such as Rho GTPases, have been identified, the dynamic cytoskeletal remodeling of specific receptor-mediated phagocytic events remain unclear. Four decades ago, two distinct mechanisms of phagocytosis, exemplified by Fc&#947; receptor (Fc&#947;R)- and complement receptor (CR)-mediated phagocytosis, were identified using scanning electron microscopy. Binding of immunoglobulin G (IgG)-opsonized particles to Fc&#947;Rs triggers the protrusion of thin membrane extensions, which initially form a so-called phagocytic cup around the particle before it becomes completely enclosed and retracted into the cell. In contrast, complement opsonized particles appear to sink into the phagocyte following binding to complement receptors. These two modes of phagocytosis, phagocytic cup formation and sinking in, have become well established in the literature. However, the distinctions between the two modes have become blurred by reports that complement receptor-mediated phagocytosis may induce various membrane protrusions. With the availability of high resolution imaging techniques, phagocytosis assays are required that allow real-time 3D (three dimensional) visualization of how specific phagocytic receptors mediate the uptake of individual particles. More commonly used approaches for the study of phagocytosis, such as end-point assays, miss the opportunity to understand what is happening at the interface of particles and phagocytes. Here we describe phagocytic assays, using time-lapse spinning disk confocal microscopy, that allow 3D imaging of single phagocytic events. In addition, we describe assays to unambiguously image Fc&#947; receptor- or complement receptor-mediated phagocytosis.

Here we describe methods using spinning disk confocal microscopy to image single phagocytic events by mouse resident peritoneal macrophages. The protocols can be extended to other phagocytic cells.

小鼠巨噬细胞吞噬的延时3D 成像

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of ...