这项工作是由美国国立卫生研究院资助AI56168，AI71142，AI87541和AI100631到MDP支持。莫里吉奥德尔Poeta的是宝来惠康研究员在传染病。

J774A.1细胞MβCD1.胆固醇消耗<ol><li>在无菌生物安全柜，种子10 5 J774A.1巨噬细胞样细胞每孔在96孔细胞培养板中加入200μlDulbecco氏改良的Eagle培养基（DMEM）中的补充有10％胎牛血清（FBS）和1％的青霉素/链霉素（P / S）。在37℃和5％CO 2的O / N。 </li><li>从细胞单层除去介质并用1×磷酸盐缓冲盐水（PBS）中已被过滤或高压灭菌洗细胞两次。 </li><li>加入200微升MβCD溶液在所需浓度（10毫米或30毫米的PBS）或1x PBS作为对照，孵育30分钟，在37℃振荡。与市售试剂盒中取出上清液和储备在室温进行定量分析，立即过程如下。 </li><li>用1×PBS或无血清DMEM洗涤细胞两到三倍，并继续与感染或裂解细胞通过pipetti纳克的两到三倍，用去离子H 2 O代表的分析用薄层色谱或用的试剂盒。 注：按照胆固醇耗竭市售胆固醇定量试剂盒可以使用。详情请参阅材料部分。按照制造商的说明进行写入。 </li></ol> 2.观察胆固醇含量薄层色谱（TLC） <ol><li> （体积比1 65：30），用石油醚中的溶液洗TLC罐两次用丙酮和一次：：乙醚乙酸。饱和罐用石油醚：乙醚：乙酸（65：30：1，体积比）溶液并离开O / N。 注：有机溶剂应始终在通风橱中使用，防止吸入蒸汽。手套和白大褂应该在任何时候都可以穿。乙酸是一种强酸，应谨慎使用。 </li><li>在无菌生物安全柜，种子10 6 J774A.1巨噬细胞样，每孔的细胞在6孔策LL培养板在5毫升温暖的DMEM补充有10％FBS和1％P / S的体积。孵育在37℃，5％的CO 2 O / N。 </li><li> 1.4，200微升，其中适用于占较大以及大小代1毫升 - 通过以下步骤1.2耗尽胆固醇的巨噬细胞。 </li><li>加入500μl胰蛋白酶EDTA至各孔中，孵育3分钟，在37℃，轻轻刮去细胞用细胞刮刀破碎。 </li><li>转移到一个离心管中，并添加另外500微升温暖的DMEM补充有10％FBS和1％P / S的。 </li><li>旋转细胞5分钟，在300×g离心并去除上清。 </li><li>增加一个额外的500微升温暖的DMEM补充有10％FBS和1％P / S的细胞沉淀，并通过上下抽吸小心重悬。 </li><li>去除10微升细胞，并在血细胞计数器计数细胞。正常化从每个样品中的细胞浓度和添加相等数目细胞的玻璃管。 注：在这一步骤中，可以选择组合的细胞来自相同的治疗组，以获得更集中的最终脂质提取物。 </li><li>离心机细胞在300 XG 5分钟在室温和删除介质。 </li><li>加入2毫升的甲醇和漩涡。然后，加入1毫升氯仿并涡旋。检查相状态，以确保在单相的解决方案。 注：管可存放在4°CO / N。 </li><li>离心在1700 xg离心在RT 10分钟，将上清转移到新的管</li><li>添加额外的1毫升氯仿接着1毫升卫生署2 O的涡旋两次30秒。离心在1700×g离心5分钟，在室温。 </li><li>权衡上的敏感平衡的玻璃管，并使用玻璃巴斯德吸管向下层相转移到已知重量的玻璃管中。 </li><li>干倒在离心蒸发器脂类至干（约2小时）。称取管干血脂，并计算干脂肪重量。 注：干脂质可以储存在-20℃，直至准备好执行TLC。 </li><li>稀干燥脂类在足够氯仿正常化脂质的浓度（通常为20 - 50微升）和负载20微升在硅胶TLC板上稀释的脂质。负载20胆固醇稀释在20μl的氯仿作为标准的微克。 </li><li>加入干薄层板上，以饱和TLC坦克，并允许溶剂可达1厘米迁移板边之前。从罐中取出TLC板，并让其干燥约5分钟。 </li><li>通过放置在碘蒸气罐，检查移民可视化脂类。删除并允许斑点褪色约10 - 引擎盖下15分钟。 注：碘是吸入危害。在通风橱中始终使用。 </li><li>通过组合60mL甲醇用60ml去离子H 2 O为4毫升硫酸，和630毫克的氯化锰制备用于中性脂质染色的溶液。 </li><li>小心缓慢地浸TLC板变成一个托盘中的中性脂肪染色解决方案，并删除不塌掉二氧化硅升艾尔。 注：中性脂质染色溶液可以重复使用好几次，因此它可以从托盘进行检索和放回瓶供以后使用。 </li><li>允许板引擎盖下干在室温，直到所有的泡沫已经消失了。加热TLC板上加热块设定为160℃和焦炭到所需的颜色。 注：光密度测定程序，如视觉作品LS可以用来量化烧焦频带进行比较的脂质样品。 </li></ol> 3.感染C.巨噬细胞新型隐球菌 （H99） <ol><li>在无菌生物安全柜，每孔在96孔细胞培养板在200μlDMEM中的种子10 5巨噬细胞样细胞在补充有10％FBS和1％P / S。孵育在37℃，5％CO 2，5％的CO 2 O / N。 注：感染，也可在玻璃完成有底为了便于成像的共焦碟子;所有金额保持不变。 </li><li> C.成长的文化新型隐球菌 （H99）接种10毫升YNB与从击打板获得的一个菌落，并与摇动孵育它O / N在30℃。 </li><li>洗和计数C.新型隐球菌 （H99）细胞。 <ol><li>离心机C.新型隐球菌 O / N培养在1700 XG为4℃10分钟。 </li><li>取出介质并丢弃。用5ml的1×PBS洗细胞。离心机在1700×g离心在4℃下10分钟。 </li><li>除去PBS中，洗涤用5ml过滤的1×PBS。离心机在1700×g离心在4℃下10分钟。重复此步骤2次以上。 </li><li>除去PBS，重悬在5毫升1X PBS的。 </li><li>使在PBS中系列稀释，得到1：500稀释洗涤培养物。 <ol><li>添加100微升原始样品至900微升的1×PBS，得到1:10稀释。 </li><li>加入100微升1时10稀释样品至900微升1×PBS中，得到1：100的稀释。 </li><li>加入200微升1：100稀释的样品到800微升1×PBS中，得到1：500 diluti上</li></ol></li><li>取10微升的1:500稀释并指望血球计算细胞的数目。 </li></ol></li><li>准备工作液激活巨噬细胞和opsonizing C.新型隐球菌。 <ol><li>加入10微升至990微升稀释LPS和IFNγ100倍的股票解决方案。 注：LPS和IFNγ用于增强巨噬细胞吞噬，但不是必须为吞噬作用。如果有兴趣的巨噬细胞的杀真菌活性，执行激活O / N在37℃下与在感染前晃动。 </li><li>每个样品结合7.5微升稀释毒素，1.25微升稀释IFNγ，1.25微升GXM抗体和C的量隐球菌培养，让1.25×10 5个细胞。带体积达到250微升乘以用DMEM样品补充有10％FBS和1％P / S的数量。 </li><li>涡旋孵育溶液中20分钟，在37℃振荡。 注意：胆固醇耗竭（步骤1.2 - 1.4）可以同时与调理步骤来完成。一定要善待之前，结合工作液的巨噬细胞，作为调理细胞应优化使用时间不超过20分钟以下步骤3.4.3孵化。 </li></ol></li><li>巨噬细胞感染<ol><li>巨噬细胞洗两次无血清DMEM，并添加200微升调理的C.隐球菌工作液到每个孔中。 </li><li>孵育2小时，在37℃。 </li></ol></li><li>修复和细胞染色<ol><li>删除媒体和DMEM洗涤细胞2次。 </li><li>空气干燥该细胞单层10分钟，并加入200微升冰冷的甲醇中以固定细胞。 </li><li>孵育15分钟，在RT和除去任何剩余的甲醇。 </li><li>加入200微升10×吉姆萨孵育5分钟，在室温。 </li><li>用去离子水和干O / N 3次盖帽关 - 洗2。 注：成像可以做到第二天或最多一周followi吴染色。 </li></ol></li><li>可视化和计数<ol><li>使用显微镜，计数每个数据点300细胞（如果有相同的治疗2计数150每孔），并注意感染的巨噬细胞的吞噬隐球菌细胞的数目和次数。 注：为了确保甚至每个条件采样每个数据点使用2片，选3每盘不重叠的区域和计数单位面积50个细胞。 </li><li>通过C的平均数乘以受感染的巨噬细胞的百分比计算吞噬指数每巨噬细胞隐球菌 。通过表达值作为1×PBS中的百分比处理的对照正常化吞噬指数。经过多次试验计算平均值和平均值的标准偏差，以确定在吞噬指数趋势。使用学生t检验，以确定显着性。 </li><li>就拿细胞的显微照片在1,000倍或400倍的放大倍率。 </li></ol></li></ol> 4.台盼蓝测定<ol><li>在无菌生物安全柜，种子10 6巨噬细胞样细胞，每孔在6孔细胞培养板中的体积5mL的Dulbecco补充有10％胎牛血清和1％青霉素/链霉素最小基本培养基（DMEM）中的。孵育在37℃，5％的CO 2 O / N。 </li><li> 1.4为200μl的在适用于占较大以及大小代2毫升 - 通过以下步骤1.2耗尽胆固醇的巨噬细胞。 注：一定要包括与1X PBS以及之前的任何处理被刮掉控制处理的对照。 </li><li>加入500微升的1×PBS的每个孔中，轻轻刮去细胞用细胞刮刀破碎。转移到一个离心管中，并轻轻吹打上下悬浮细胞。 </li><li>去除10微升细胞和染色用1微升4％台盼蓝。 </li><li>计数细胞在血细胞计数器，并使用以下等式计算存活率：％存活率= [1 - （蓝色细胞/总细胞）]×100标准化值，这是未经处理的控制。经过多次试验计算的平均值和标准偏差，以确定在存活力的趋势。使用学生t检验，以确定显着性。 </li></ol>

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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydroxyurea+Treatment+Inhibits+Proliferation+Of+Cryptococcus+Neoformans+In+Mice.">Hydroxyurea Treatment Inhibits Proliferation Of Cryptococcus Neoformans In Mice.</a> Frontiers In Microbiology. 3, 187 (2012).</li><li>Chaka, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Analysis+Of+Phagocytosis+And+Killing+Of+Cryptococcus+Neoformans+By+Human+Peripheral+Blood+Mononuclear+Cells+By+Flow+Cytometry.">Quantitative Analysis Of Phagocytosis And Killing Of Cryptococcus Neoformans By Human Peripheral Blood Mononuclear Cells By Flow Cytometry.</a> Clin. Diagn. Lab. Immunol. 2 (6), 753-759 (1995).</li></ol>

The authors have nothing to disclose.

在这个协议的工作是电镀时哺乳动物细胞和opsonizing C.获得准确的细胞计数是非常重要的新型隐球菌细胞。该试验间差异最小化，并保证准确的1：整个研究1目标效应比。这也是至关重要的协调胆固醇枯竭和感染的时间，以防止调理的酵母细胞或巨噬细胞处理细胞在RT中的过程之间的休息。漫长的等待时间可能会导致损失抗体的调理作用或贫胆固醇补给之前感染就可以开始。如果实验?...

吞噬作用是由细胞外的实体是由宿主细胞内化的过程。它是在免疫系统中的武器库，以对抗病原体保卫一个关键武器，但该过程可以经常由病原体被颠覆，以允许内化和整个主体1传播。吞噬作用是由导致通过宿主细胞的细胞骨架的重排附着和吞噬几个信号传导事件介导的。职业“吞噬细胞能够识别和结合调理素的入侵病原体的表面上的信号用于附着和形成片状伪足的，其吞噬的病原体，并形成吞噬体2。其中所谓的&#39;专业&#39;吞噬细胞巨噬细胞是。巨噬细胞是高度特化的细胞即进行保护的功能包括寻找和消除致病剂，修复受损的组织，并介导的炎症，大多数的这些通过吞噬1,2的过程。 新生隐球菌是一个物种致病酵母导致称为隐球菌一个严重的疾病。 隐球菌孢子由主机吸入并导致肺部感染，通常是无症状的。据认为，曝光是极为普遍;对61名儿童从儿科传染病诊所在布朗克斯-黎巴嫩医院中心发现，所有的受访者有抗体的隐球菌多糖glucuronoxylomannan和其他研究的样本表明患病率在人类免疫缺陷病毒（HIV）感染和感染的成人3， 4。肺泡巨噬细胞是响应于肺部感染，并且在大多数情况下，成功地明确病原体的第一道防线。然而，在免疫受损的个体（ 例如，HIV和AIDS患者）的酵母是能够在巨噬细胞内生存。在这些的情况下，巨噬细胞可以作为利基病原体的复制，并且可以促进其传 ​​播到中枢神经系统（CNS），其中该疾病成为致命5 - 8。据认为，巨噬细胞可能甚至直接传递到酵母脑膜，帮助酵母通过“木马”的模式3,9穿过血脑屏障- 11。因此，为了理解吞噬的过程和影响它，特别是在隐球菌感染的因素是很重要的。 以前的工作在其它病原体系统指向由胆固醇作为具有重要作用，吞噬12发挥形成胆固醇和脂质筏- 15。胆固醇是最丰富的脂类物质在哺乳动物细胞中，并包括25 -哺乳动物细胞膜16的50％。已经发现，以在调节BIOP发挥作用膜通过改变其刚性物质环境的17性质。胆固醇和鞘脂在一起形成被称为脂筏在膜中的脂质微区。脂筏已经发现涉及在地层中的小窝，以及提供一个分离的结构域对某些类型的信号16 - 18。由于它们的尺寸小，它是很难研究脂质筏体内 。研究脂筏的作用的一个有用的方法是改变他们的选民。甲基β环糊精（MβCD）是已被发现从哺乳动物细胞膜耗尽胆固醇，通常用于研究脂质筏18的作用的化合物。 在这个协议中，我们提出了一个方法，以消耗从宿主细胞膜胆固醇并量化对宿主细胞的吞噬C的能力的耗尽的效果隐球菌体外 。此过程使用了细胞培养techniqUE的像细胞（J774A.1）作为模型感染的巨噬细胞的永生。胆固醇耗竭被完成通过暴露于MβCD，其具有特定的甾醇的尺寸的疏水性芯，并且能够充当胆固醇绘制出来的膜19的一个接收器。定量用市售试剂盒和定性使用改良的Bligh-Dyer的脂质提取，随后用薄层色谱法（TLC）20胆固醇耗竭进行测定。 23 -吞噬被感染与调理的酵母与干扰素γ和脂多糖的鸡尾酒激活巨噬细胞混合培养的细胞系测量隐球菌是使用glucuronoxylomannan（GXM）抗体21调理。染色和显微镜实验允许的细胞可视化和吞噬指数的计算，以评估细胞吞噬的程度。综上所述，该协议ðescribes集成脂质组合物具有的生理过程的改变的基本方法。

<table border="0" cellpadding="0" cellspacing="0" width="622">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name of Reagent/ Equipment</td>
			<td style="width:120px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Class II type A2 Biosafety Cabinet</td>
			<td style="width:120px;">Labconco</td>
			<td style="width:119px;">3460009</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">J774A.1 cell line</td>
			<td style="width:120px;">ATCC</td>
			<td style="width:119px;">TIB-67</td>
			<td style="width:167px;">Arrives Frozen. See ATCC instructions for culturing.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dulbecco&#8217;s Modified Eagle Medium</td>
			<td style="width:120px;">Gibco</td>
			<td style="width:119px;">11995-065</td>
			<td style="width:167px;">Store at 4 &#176;C and warm to 37 &#176;C prior to use</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">HI Fetal Bovine Serum Performance Plus</td>
			<td style="width:120px;">Gibco</td>
			<td style="width:119px;">10082-147</td>
			<td style="width:167px;">Keep frozen at -20 &#176;C and thaw before adding to DMEM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:120px;">Gibco</td>
			<td style="width:119px;">15140-122</td>
			<td style="width:167px;">Used to suplement DMEM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isotemp Cell culture incubator</td>
			<td style="width:120px;">Fisher Scientific</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Model # 3530</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">96-Well culture dish</td>
			<td style="width:120px;">Corning Inc. Costar</td>
			<td style="width:119px;">3595</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">10x Phosphate Buffered Saline</td>
			<td style="width:120px;">Fisher Scientific BioReagents</td>
			<td style="width:119px;">BP3994</td>
			<td style="width:167px;">Dilute to 1x and filter or autoclave prior to use.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Methyl-&#946;-Cyclodextrin</td>
			<td style="width:120px;">Sigma Life Science</td>
			<td style="width:119px;">C4555-10G</td>
			<td style="width:167px;">Dissolve in 1x PBS to make solutions of 10mM and 30mM concentrations</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Orbital Shaker</td>
			<td style="width:120px;">Labline</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Amplex Red Cholesterol Assay Kit</td>
			<td style="width:120px;">Life Technologies Molecular Probes</td>
			<td style="width:119px;">A12216</td>
			<td style="width:167px;">All reagents for Cholesterol Assay are contained within the kit. Follow Manufacturer instructions.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">96-Well Black Assay plate</td>
			<td style="width:120px;">Corning Inc. Costar</td>
			<td style="width:119px;">3603</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">FilterMax microplate reader</td>
			<td style="width:120px;">Molecular Devices</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Model F5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TLC Chamber</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z126195-1EA</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Chloroform</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:119px;">650498-4L</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Methanol</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:119px;">34860-2L-R</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TLC Paper</td>
			<td style="width:120px;">Whatman</td>
			<td style="width:119px;">3030917</td>
			<td style="width:167px;">Cut down to size needed for TLC tank</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Fume Hood</td>
			<td style="width:120px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Any fume hood that complies with AIHA/ANSI Standards&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">6-Well Plate</td>
			<td style="width:120px;">Corning Inc. Costar</td>
			<td style="width:119px;">3506</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin-EDTA</td>
			<td style="width:120px;">Gibco</td>
			<td style="width:119px;">25300-054</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cell Scraper</td>
			<td style="width:120px;">Corning Inc. Costar</td>
			<td style="width:119px;">3010</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hemocytometer&#160;</td>
			<td style="width:120px;">Hausser Scientific</td>
			<td style="width:119px;">1490</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifuge</td>
			<td style="width:120px;">Beckman Coulter</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Model Alegra x-30R</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Votex Mixer</td>
			<td style="width:120px;">Fisher Scientific</td>
			<td style="width:119px;">12-812</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Balance</td>
			<td style="width:120px;">Mettler Toledo</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Model # MS104S Meaures down to .1 mg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass Pasteur Pipette</td>
			<td style="width:120px;">Fisherbrand</td>
			<td style="width:119px;">13-678-20A</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cholesterol</td>
			<td style="width:120px;">Avanti Polar Lipids</td>
			<td style="width:119px;">700000</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SpeedVac Concentrator</td>
			<td style="width:120px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Model # SPD2010&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Petroleum Ether</td>
			<td style="width:120px;">Fisher Scientific</td>
			<td style="width:119px;">E139-1&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Diethyl Ether</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:119px;">309966&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Acetic Acid</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:119px;">320099</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">TLC Silica Gel 60 with concentrating zone</td>
			<td style="width:120px;">Analytical Chromatograhy Millipore</td>
			<td style="width:119px;">1.11845.0001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Iodine Chips</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:119px;">376558-50G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sulfuric Acid</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:119px;">320501&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Manganese Chloride</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:119px;">244589</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">UVP EC3 Imaging System</td>
			<td style="width:120px;">Ultra-Violet Products Ltd.</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Use the Vision Works LS software for densitometry analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass Bottom Confocal Dish</td>
			<td style="width:120px;">MatTek</td>
			<td style="width:119px;">P35G-1.5-10C</td>
			<td>www.glassbottomdishes.com</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Cryptococcus neoformans (H99)</td>
			<td style="width:120px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Obtained from Duke University Medical Center</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">YNB</td>
			<td style="width:120px;">BD</td>
			<td style="width:119px;">239210</td>
			<td style="width:167px;">See manufacturer for preparation instructions. Use a Glucose concentration of 20 g/L.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Lipopolysaccharide</td>
			<td style="width:120px;">Sigma</td>
			<td style="width:119px;">L4391-1MG</td>
			<td style="width:167px;">Dissolve in 1x PBS to make 1mg/mL stock. Store at -20 &#176;C.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Interferon gamma</td>
			<td style="width:120px;">Sigma</td>
			<td style="width:119px;">I4777</td>
			<td style="width:167px;">Dissolve in 1x PBS to make .1 mg/mL stock solution</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Glucuronoxylomannan antibody (anti-GXM)</td>
			<td style="width:120px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Gift from Arturo Casadevall&#39;s Lab concentration is 1.98 mg/mL</td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:216px;">Giemsa</td>
			<td style="width:120px;">MP Biomedicals</td>
			<td style="width:119px;">194591</td>
			<td style="width:167px;">Dissolve .8 g of Giemsa in 25 mL of Glycerol and heat to 60 &#176;C for 1 hour. Add 25 mL of methanol to the solution and allow to age at room temperature for at least 1 month.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Microscope</td>
			<td style="width:120px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Observer.D1 microscope with AxioCam MRm for taking images</td>
		</tr>
	</tbody>
</table>

macrophage cholesterol depletion and its effect on the phagocytosis of cryptococcus neoformans

隐球菌是一种威胁生命的感染引起的属隐球菌的致病性真菌。发生感染时吸入孢子，这是能够在肺部深处复制的。 隐球菌的巨噬细胞吞噬是，该疾病是能够传播到中枢神经系统以引起致死性脑膜脑炎的方法之一。因此， 隐球菌和巨噬细胞之间的关联的研究是非常重要的，以了解感染的进展。本研究描述了一个一步一步的协议，由C.研究巨噬细胞感染隐球菌体外 。使用该协议，主机固醇对宿主 - 病原体相互作用的作用进行了研究。不同浓度的甲基 - 环糊精（MCD）用于消耗来自鼠网肉瘤巨噬细胞样细胞系J774A.1胆固醇。胆固醇耗竭进行确认和定量同时使用市售availablË胆固醇定量试剂盒和薄层色谱法。用脂多糖（LPS）和干扰素γ（IFNγ）胆固醇耗竭细胞被激活，并感染抗体调理隐球菌野生型H99细胞以1的效应-目标：1的比例。 2小时温育与C之后感染的细胞进行了监测新型隐球菌及吞噬指数计算。胆固醇耗竭导致一显著降低的吞噬指数。所提出的协议，提供了一个方便的方法，以模仿感染过程的开始在实验室环境中，研究宿主脂质组合物对感染性的作用。

胆固醇消耗相比于1×PBS对照由以下中的Amplex红胆固醇测定试剂盒制造商的说明在协议的步骤1.3保留上清液的分析产生一个升高的浓度的胆固醇在MβCD处理过的样品。取决于细胞类型和MβCD浓度使用胆固醇耗竭可能会发生变化。对于J774用10mMMβCD处理后，观察到大约50％的损耗。耗尽可以使用从收集在步骤1.4（ 图1）的上清液和细胞裂解物获得的值来计算。 <p class="jove...

Watch this Scientific Journal Video about Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans at JoVE.com

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

In this article, a protocol for infection of macrophages with Cryptococcus neoformans is described. Also, a method for sterol depletion from the macrophages is explained. These protocols provide a guide to study fungal infections in vitro and examine the role of sterols in such infections.

巨噬细胞胆固醇消耗及其对的吞噬作用<em&gt;新型隐球菌</em

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are ...

macrophage-cholesterol-depletion-its-effect-on-phagocytosis

Research

JoVE Journal

Immunology and Infection

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

12.6K 次观看.  Stony Brook University. In this article, a protocol for infection of macrophages with Cryptococcus neoformans is described. Also, a method for sterol depletion from the macrophages is explained. These protocols provide a guide to study fungal infections in vitro and examine the role of sterols in such infections. 

视频: Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37&deg;C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

补消耗的特定细胞群的枯竭

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

科研

免疫与感染

Depletion and Reconstitution of Macrophages in Mice

Macrophages are critical players in the innate immune response to infectious challenge or injury, initiating the innate immune response and directing the acquired immune response. Macrophage dysfunction can lead to an inability to mount an appropriate immune response and as such, has been implicated in many disease processes, including inflammatory bowel diseases. Macrophages display polarized phenotypes that are broadly divided into two categories. Classically activated macrophages, activated by stimulation with IFN&gamma; or LPS, play an essential role in response to bacterial challenge whereas alternatively activated macrophages, activated by IL-4 or IL-13, participate in debris scavenging and tissue remodeling and have been implicated in the resolution phase of inflammation. During an inflammatory response in vivo, macrophages are found amid a complex mixture of infiltrating immune cells and may participate by exacerbating or resolving inflammation. To define the role of macrophages in situ in a whole animal model, it is necessary to examine the effect of depleting macrophages from the complex environment. To ask questions about the role of macrophage phenotype in situ, phenotypically defined polarized macrophages can be derived ex vivo, from bone marrow aspirates and added back to mice, with or without prior depletion of macrophages. In the protocol presented here clodronate-containing liposomes, versus PBS injected controls, were used to deplete colonic macrophages during dextran sodium sulfate (DSS)-induced colitis in mice. In addition, polarized macrophages were derived ex vivo and transferred to mice by intravenous injection. A caveat to this approach is that clodronate-containing liposomes deplete all professional phagocytes, including both dendritic cells and macrophages so to ensure the effect observed by depletion is macrophage-specific, reconstitution of phenotype by adoptive transfer of macrophages is necessary. Systemic macrophage depletion in mice can also be achieved by backcrossing mice onto a CD11b-DTR background, which is an excellent complementary approach. The advantage of clodronate-containing liposome-mediated depletion is that it does not require the time and expense involved in backcrossing mice and it can be used in mice regardless of the background of the mice (C57BL/6, BALB/c, or mixed background).

Macrophages play a central role in homeostasis and pathology in many tissues. The protocol presented here describes methods for depleting macrophages in vivo, deriving polarized macrophages from bone marrow aspirates, and adoptively transferring macrophages into mice. These techniques allow determination of the role that polarized macrophages play in health and disease.

在小鼠体内的巨噬细胞的枯竭和重建

Macrophages are critical players in the innate  immune response to infectious challenge or injury, initiating the innate immune  response and directing ...

The Synergistic Effect of Visible Light and Gentamycin on Pseudomona aeruginosa Microorganisms

Recently there were several publications on the bactericidal effect of visible light, most of them claiming that blue part of the spectrum (400 nm-500 nm) is responsible for killing various pathogens1-5. The phototoxic effect of blue light was suggested to be a result of light-induced reactive oxygen species (ROS) formation by endogenous bacterial photosensitizers which mostly absorb light in the blue region4,6,7. There are also reports of biocidal effect of red and near infra red8 as well as green light9.
In the present study, we developed a method that allowed us to characterize the effect of high power green (wavelength of 532 nm) continuous (CW) and pulsed Q-switched (Q-S) light on Pseudomonas aeruginosa. Using this method we also studied the effect of green light combined with antibiotic treatment (gentamycin) on the bacteria viability. P. aeruginosa is a common noscomial opportunistic pathogen causing various diseases. The strain is fairly resistant to various antibiotics and contains many predicted AcrB/Mex-type RND multidrug efflux systems10.
The method utilized free-living stationary phase Gram-negative bacteria (P. aeruginosa strain PAO1), grown in Luria Broth (LB) medium exposed to Q-switched and/or CW lasers with and without the addition of the antibiotic gentamycin. Cell viability was determined at different time points. The obtained results showed that laser treatment alone did not reduce cell viability compared to untreated control and that gentamycin treatment alone only resulted in a 0.5 log reduction in the viable count for P. aeruginosa. The combined laser and gentamycin treatment, however, resulted in a synergistic effect and the viability of P. aeruginosa was reduced by 8 log's.
The proposed method can further be implemented via the development of catheter like device capable of injecting an antibiotic solution into the infected organ while simultaneously illuminating the area with light.

The Synergistic Effect of Visible Light and Gentamycin on Pseudomona aeruginosa Microorganisms

We show that a developed biomedical device involving continuous or pulsed visible laser based treatment that is combined with antibiotic treatment (gentamycin), results in a statistically significant synergistic effect leading to a reduction in the viability of P. aeruginosa PAO1, by 8 log's compared to antibiotic treatment alone.

可见光和庆大霉素的协同作用<em&gt;假单胞菌假单胞菌</em&gt;微生物

Recently there were several publications on the  bactericidal effect of visible light, most of them claiming that blue part of  the spectrum (400 nm-500 ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

使用点击化学测量的影响，病毒感染宿主细胞RNA合成

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

Many aspects of the infection of macrophages by Cryptococcus neoformans have been extensively studied and well defined. However, one particular interaction that is not clearly understood is non-lytic exocytosis. In this process, yeast cells are released into the extracellular space by a poorly understood mechanism that leaves both the macrophage and Cn viable. Here, we describe how to follow a large number of individually infected macrophages for a 24 hr infection period by time-lapsed microscopy. Infected macrophages are housed in a heating chamber with a CO2 atmosphere attached to a microscope that provides the same conditions as a cell-culture incubator. Live digital microscopy can provide information about the dynamic interactions between a host and pathogen that is not available from static images. Being able to visualize each infected cell can provide clues as to how macrophages handle fungal infections, and vice versa. This technique is a powerful tool in studying the dynamics that are behind a complex phenomenon.

Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

We describe how to visualize macrophage-C. neoformans (Cn) interactions in real time, with specific emphasis on the process of non-lytic exocytosis using digital light microscopy. Using this technique individually infected macrophages can be studied to ascertain various aspects of this phenomenon.

可视化的非溶胞胞吐作用<em&gt;新型隐球菌</em&gt;从巨噬细胞用数码光学显微镜

Many aspects of the infection of macrophages by Cryptococcus neoformans have been extensively studied and well defined. However, one particular ...

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular processes. This technique can be used on a variety of cell types, both adherent and non-adherent, to examine a variety of cellular properties. When studying phagocytosis, fluorometric technique utilizes phagocytic cell types such as macrophages, and fluorescently labeled opsonized particles whose fluorescence can be extinguished in the presence of trypan blue. Following plating of adherent macrophages in 96-well plates, fluorescent particles (green or red) are administered and cells are allowed to phagocytose for varied amounts of time. Following internalization of fluorescent particles, cells are washed with trypan blue, which facilitates extinction of fluorescent signal from bacteria which are not internalized, or are merely adhering to the cell surface. Following the trypan wash, cells are washed with PBS, fixed, and stained with DAPI (nuclear blue fluorescent label), which serves to label nuclei of cells. By a simple fluorometric quantification through plate reading of nuclear (blue) or particle (red/green) fluorescence we can examine the ratio of relative fluorescence units of green:blue and determine a phagocytic index indicative of amount of fluorescent bacteria internalized per cell. The duration of assay using a 96-well method and multichannel pipettes for washing, from end of phagocytosis to end of data acquisition, is less than 45 min. Flow cytometry could be used in a similar manner but the advantage of fluorometry is its high throughput, rapid method of assessment with minimal manipulation of samples and quick quantification of fluorescent intensity per cell. Similar strategies can be applied to non adherent cells, live labeled bacteria, actin polymerization, and essentially any process utilizing fluorescence. Therefore, fluorometry is a promising method for its low cost, high throughput capabilities in the study of cellular processes.

Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.

高通量荧光技术巨噬细胞吞噬功能和肌动蛋白聚合的评估

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular ...

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal meningitis worldwide resulting in more than 625,000 deaths per year worldwide.&#160;Although electroporation has been developed for the transformation of plasmids in Cryptococcus, only&#160;biolistic&#160;delivery provides&#160;an effective means to transform&#160;linear&#160;DNA&#160;that can be integrated into the genome by homologous recombination.&#160;
Acetate has been shown to be a major fermentation product during cryptococcal infection, but the significance of this is not yet known.&#160;A bacterial pathway composed of the enzymes xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp) and acetate kinase (Ack) is one of three potential pathways for acetate production in C. neoformans. Here, we demonstrate the biolistic transformation of a construct, which has the gene encoding Ack fused to the fluorescent tag mCherry, into&#160;C. neoformans. We then confirm integration of the ACK-mCherry fusion into the&#160;ACK&#160;locus.

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

Biolistic transformation is a method used to generate stable integration of DNA into the genome of the opportunistic pathogen Cryptococcus neoformans through homologous recombination. We will demonstrate biolistic transformation of a construct, which has the gene encoding acetate kinase fused to the fluorescent tag mCherry into&#160;C. neoformans.

荧光标记基因的基因枪转化成机会性真菌病原<em&gt;新型隐球菌</em

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal ...

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. However, isolating bacterial RNA from infected cells or tissues is a challenging task, since mammalian RNA mostly dominates the lysates of infected cells. Here we describe an optimized method for RNA isolation of Listeria monocytogenes bacteria growing within bone marrow derived macrophage cells. Upon infection, cells are mildly lysed and rapidly filtered to discard most of the host proteins and RNA, while retaining intact bacteria. Next, bacterial RNA is isolated using hot phenol-SDS extraction followed by DNase treatment. The extracted RNA is suitable for gene transcription analysis by multiple techniques. This method is successfully employed in our studies of Listeria monocytogenes gene regulation during infection of macrophage cells 1-4. The protocol can be easily modified to study other bacterial pathogens and cell types.

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Here we describe a method for bacterial RNA isolation from Listeria monocytogenes bacteria growing inside murine macrophages. This technique can be used with other intracellular pathogens and mammalian host cells.

RNA纯化在细胞内生长<em&gt;李斯特菌</em&gt;巨噬细胞

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. ...

Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages are equally important in turning off inflammatory responses. We have shown that macrophages stimulated with intravenous immunoglobulin (IVIg) can produce high amounts of the anti-inflammatory cytokine, interleukin 10 (IL-10), and low levels of pro-inflammatory cytokines in response to bacterial lipopolysaccharides (LPS). IVIg is a polyvalent antibody, primarily immunoglobulin Gs (IgGs), pooled from the plasma of more than 1,000 blood donors. It is used to supplement antibodies in patients with immune deficiencies or to suppress immune responses in patients with autoimmune or inflammatory conditions. Infliximab, a therapeutic anti-tumor necrosis factor alpha (TNF&#945;) antibody, has also been shown to activate macrophages to produce IL-10 in response to inflammatory stimuli. IVIg and other antibody-based biologics can be tested to determine their effects on macrophage activation. This paper describes methods for derivation, stimulation, and assessment of murine bone marrow macrophages activated by antibodies in vitro and murine peritoneal macrophages activated with antibodies in vivo. Finally, we demonstrate the use of western blotting to determine the contribution of specific cell signaling pathways to anti-inflammatory macrophage activity. These protocols can be used with genetically modified mice, to determine the effect of a specific protein(s) on anti-inflammatory macrophage activation. These techniques can also be used to assess whether specific biologics may act by changing macrophages to an IL-10-producing anti-inflammatory activation state that reduces inflammatory responses in vivo. This can provide information on the role of macrophage activation in the efficacy of biologics during disease models in mice, and provide insight into a potential new mechanism of action in people. Conversely, this may caution against the use of specific antibody-based biologics to treat infectious disease, particularly if macrophages play an important role in host defense against that infection.

Antibody-based drugs have revolutionized treatment for inflammatory diseases. In addition to having direct effects on specific targets, antibodies can activate macrophages to become anti-inflammatory. This protocol describes how anti-inflammatory macrophage activation can be assessed in vitro, using mouse bone marrow macrophages, and in vivo, using peritoneal macrophages.

抗体药物对小鼠骨髓及腹腔巨噬细胞活化作用的评价

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages ...

Size Matters: Measurement of Capsule Diameter in Cryptococcus neoformans

The polysaccharide capsule of Cryptococcus neoformans is the primary virulence factor and one of the most commonly studied aspects of this pathogenic yeast. Capsule size can vary widely between strains, has the ability to grow rapidly when introduced to stressful or low nutrient conditions, and has been positively correlated with strain virulence. For these reasons, the size of the capsule is of great interest to C. neoformans researchers. The growth of the C. neoformans capsule is induced during phenotypic testing to help understand the effects of different treatments on the yeast or size differences between strains. Here we describe one of the standard methods of capsule induction and compare two accepted methods of staining and measuring capsule diameter: (i) India ink, a negative stain, used in conjunction with conventional light microscopy and (ii) co-staining with fluorescent dyes of both the cell wall and capsule followed by confocal microscopy. Finally, we show how measurement of capsule diameter from India ink-stained samples can be automated using computational image analysis.

Size Matters: Measurement of Capsule Diameter in Cryptococcus neoformans

The polysaccharide capsule is the primary virulence factor in Cryptococcus neoformans, and its size correlates with strain virulence. Capsule diameter measurements are used in phenotypic testing and to gauge therapeutic efficacy. Here a standard method of capsule induction is presented, and two methods of staining and measuring diameter are compared.

尺寸事项:新生隐球菌中胶囊直径的测定

The polysaccharide capsule of Cryptococcus neoformans is the primary virulence factor and one of the most commonly studied aspects of this pathogenic ...