Name: quantification of intracellular growth inside macrophages is a fast and reliable method for assessing the virulence of leishmania parasites
Uploaded: 2018-03-16T15:00:00.000+00:00
Duration: 10 min 1 s
Description: Watch this Scientific Journal Video about Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites at JoVE.com

这项工作得到了国家卫生研究院资助 RO1 AI067979 NWA。 YK 是霍华德休斯医学院/马里兰大学帕克分校的本科生奖学金获得者。

所有的实验程序都是按照《国家卫生研究院护理和使用实验动物指南》的建议进行的, 并得到马里兰大学 IACUC 的批准。1至4节所述的所有步骤都应在生物层流柜内灌装进行。应使用个人保护, 在处理所有试验阶段的活的利什曼原虫寄生虫时应谨慎行事。
1. 骨髓源巨噬细胞的分离和分化 (BMMs)8,30,31
<ol><li>
0 天:在 CO2室中牺牲 4-6 周大的雌性C57BL/6或BALB鼠标, 并通过颈椎脱位确认死亡。确保剪刀和镊子的灭菌, 使其浸泡在70% 乙醇。喷雾手套与70% 乙醇, 以允许无菌处理整个骨髓细胞隔离过程。</li>
	<li>通过用70% 乙醇喷洒丰富, 将其前肢固定, 并对其进行消毒。</li>
	<li>在髋关节附近用剪刀做一个小切口 (约1厘米)。小心地在皮肤下插入剪刀以分离底层肌肉, 并在髋关节周围切开皮肤。 注: 解剖板可以旋转, 以允许改善对髋关节的接触。</li>
	<li>小心 deglove 的皮肤从腿部拉向脚踝和删除。在脚踝上切下脚, 小心地在脚踝关节处切开, 使胫骨完全完好无损。 注: 在这个阶段的腓骨是松散的连接, 可以很容易地与镊子分离。</li>
	<li>通过操纵股骨来手动定位髋关节以确定旋转点。小心使用剪刀切断髋关节以上的腿, 使股骨的近端头部完好无损。</li>
	<li>用剪刀从腿部取出尽可能多的肌肉和结缔组织。从脚踝和髋关节上方的任何多余的骨材料中取出任何剩余的皮肤。</li>
	<li>安置被清洗的腿骨头在无菌 RPMI 补充青霉素或链霉素 (1% 最后的集中) 在培养皿在冰。</li>
	<li>重复步骤1.4 至 1.6, 把胫骨和股骨从另一条后腿分离出来。</li>
	<li>用灭菌钳和无菌 #10 刀片从 RPMI 浸泡的骨骼中取出残余的肌肉和结缔组织。</li>
	<li>把骨头放在培养皿盖子上。用刀片在踝关节上方仔细切开胫骨以进入骨髓。将胫骨从腿部的其余部分切除, 然后立即在膝关节下方切开。</li>
	<li>绘制10毫升无菌 RPMI 补充青霉素/链霉素 (1% 最终浓度) 成10毫升注射器和附加25G 针。</li>
	<li>用镊子牢牢地握住胫骨, 在冰上垂直超过50毫升锥形管。用注射器轻轻地注入大约5毫升 RPMI 到骨头的末端, 将骨髓从另一端冲洗成锥形管。用总共10毫升 RPMI 的骨髓冲洗, 直到骨头变成白色。 注: 彻底冲洗后, 骨应变白。如果没有, 继续冲洗, 以去除骨髓。胫骨远端可能需要修剪与刀片, 如果它是太窄, 以插入针。</li>
	<li>立即将股骨置于膝关节上方, 并立即在髋关节下方切开, 露出骨的两端以进入骨髓。用与胫骨相同的方式从股骨中冲洗骨髓, 共10毫升 RPMI。</li>
	<li>重复清洗和冲洗步骤1.6 至1.13 与其余的骨骼和收集骨髓冲洗细胞在一个50毫升锥管 注: 如果骨冲洗前在解剖过程中的任何一点骨折, 不要使用骨髓从这块骨头。</li>
	<li>离心机用于从所有四块骨头中提取骨髓的锥形管, 10 分钟4摄氏度, 300 x g。</li>
	<li>放置60毫升无菌法钢公司培养基 (RPMI 的最终浓度为 20%, 1% 青霉素/链霉素, 1.2 毫米 Na-丙酮酸, 25 µg/毫升人类脑脊液) 成 175厘米2 细胞培养瓶与过滤帽。</li>
	<li>用5毫升的法钢公司培养基从瓶中并用重悬, 在离心后丢弃上清液, 然后用温和向上和向下吹打细胞颗粒。</li>
	<li>将细胞悬浮液转移到培养瓶中, 用法钢公司培养基从培养瓶中冲洗两次锥形管, 以保证细胞的最大回收率。确保细胞悬浮液彻底混合成培养基, 并将30毫升从最初的175厘米2细胞培养瓶中分配到第二175厘米2细胞培养瓶中, 过滤器盖。</li>
	<li>孵化两个含有30毫升细胞悬浮液的烧瓶, 每晚在37摄氏度, 5% CO2 , 以允许分离任何污染的成纤维细胞, 居民巨噬细胞, 和其他黏附体细胞。</li>
	<li>
1 天:将含有未分化骨髓细胞的上清液从烧瓶转移到100毫米 x 15 毫米非 TC 处理的聚苯乙烯培养皿, 约10毫升/碟, 孵育37°c, 5% CO2.扔掉烧瓶。</li>
	<li>
3 或4天:添加一个额外的5毫升法钢公司培养基 (预热到37°c) 到每个培养皿, 并继续孵化37°c, 5%CO2。</li>
</ol>2. 盖玻片感染的电镀 BMMs (7 或8天)
<ol><li>在每个空井的底部放置四 (4) 无菌12毫米玻璃盖玻片 (蒸压并贮存在密闭的玻璃容器中), 准备6井组织培养板。 注: 选择每一个无菌12毫米盖玻片与一个吸气尖端安装在真空线。将盖玻片放入井中, 不加重叠, 通过捏管以中断真空, 释放在所需位置。</li>
	<li>从培养皿上涂上粘附骨髓巨噬细胞的培养基 (以及任何非粘附单元), 轻轻冲洗黏附细胞2x 无菌 Dulbecco 磷酸盐缓冲盐水无钙和氯化镁 (PBS-/) 预预热到37°c。</li>
	<li>添加滴状, 5 毫升无菌 1x PBS-/与最终浓度为1毫米 EDTA 每培养皿和孵化5分钟在37°c, 以分离黏附 BMMs. 验证单元格是否使用显微镜分离。</li>
	<li>收集所有分离的细胞悬浮在 1x PBS-/补充与1毫米 EDTA 在50毫升圆锥管。用5毫升 PBS 冲洗每道菜 2x, 并加入细胞悬浮池。 注: 细胞悬浮可能被稀释与额外的 PBS--以保护巨噬细胞从 EDTA 毒性在收集过程中。</li>
	<li>离心机在 300 x g 10 分钟在4摄氏度。用吹打将上清和并用重悬巨噬细胞在 5-10 毫升法钢公司培养基中丢弃。在冰上放置悬浮细胞, 以防止附着在管子和结块。</li>
	<li>在 hemocytometer 中使用台盼蓝排除计数可行细胞。 注: 要计数法钢公司细胞悬浮在5毫升培养基中, 稀释混合25µL 细胞悬浮在445µL PBS-/和30µL 0.4% 台盼蓝解决方案通常允许简单的量化在 hemocytometer (考虑到20x 稀释因子的最终计算).仅计数没有被台盼蓝染色的细胞 (可行细胞)。如果这种混合物过于浓缩或稀释, 细胞悬浮量可能会改变。一般情况下, 每个准备法钢公司的收率在 2 x10 7 和 5 x 10 7 单元格之间从单个鼠标中变化.</li>
	<li>在所需的电镀浓度 (5 x 105/毫升) 和分散悬浮到6井板中, 在2毫升介质中准备法钢公司介质中的悬浮液, 以便每个井接收 1 x10 6 巨噬细胞。 注意: 这是一个关键步骤, 确保每一个井得到足够数量的巨噬细胞均匀地覆盖盖玻片。检查井中的盖玻片不重叠或漂浮在介质中。如果是这样, 用不育的吸管尖轻轻调整。</li>
	<li>孵化过夜在37°c, 5% CO2。</li>
</ol>3. 对感染形式的amazonensis的纯化
注意:使用标准 L 前鞭毛体从固定 promastigote区域性8、13或将区域性中的 amastigote 区分为 amazonensis 形式, 为感染-净化 metacyclic 前鞭毛体准备利什曼原虫。无菌差异协议 6, 8.
<ol><li>
使用密度梯度介质 (材料) 对利什曼原虫metacyclic 前鞭毛体进行纯化33<ol>
<li>在无菌无内毒素水中制备40% 种密度梯度介质的溶液。</li>
		<li>稀释密度梯度溶液在 10x M199 培养基 (不含血清) 中制备10% 浓度的 M199 培养基。</li>
		<li>通过0.22 µm 过滤器筛选所有解决方案。 注意: 库存解决方案可以在黑暗中存储 4 oC, 不超过1月。</li>
		<li>在15毫升锥形离心管的底部添加2毫升40% 密度梯度介质溶液。</li>
		<li>2毫升的10% 密度梯度介质溶液在 M199 上的40% 密度梯度介质层仔细, 使用巴斯德吸管, 以避免任何混合在两层之间。</li>
		<li>从固定相培养中收集 1 x 109寄生虫, 离心在 1900 x g 处以10分钟。</li>
		<li>并用重悬细胞在6毫升 Dulbecco 的修饰必需培养基 (DMEM)。</li>
		<li>2毫升的细胞悬浮在10% 密度梯度介质层的顶端, 轻轻地用巴斯德吸管避免在层间混合。</li>
		<li>在室温下, 离心机在 1300 x g 处的梯度为10分钟。 注意: 由于利什曼原虫种之间的物理特性不同, 每个可能需要稍微调整离心条件以确保最大产量。</li>
		<li>收集寄生虫 (丰富的 metacyclic promastigote 形式) 从形成在上层10% 密度梯度介质边界 (介面之间的0% 和10% 密度梯度介质层)。</li>
		<li>稀释寄生虫与一容量 DMEM 和收集通过离心 (1900 x g 10 分钟在室温)。</li>
		<li>并用重悬在500µL DMEM 培养基中, 计数 hemocytometer 以量化产量。</li>
	</ol>
</li>
	<li>
在无菌区域性条件下生成 amazonensis amastigotes (低 pH 值/高温 ) 6, 8, 13<ol>
<li>混合5毫升的对数相 promastigote 培养 (ph 7.4 在 26 oC) 与等量的 amastigote 培养基 (ph 4.5), 使用25厘米2烧瓶 (总10毫升培养基) 和孵化在26°c 过夜。</li>
		<li>将烧瓶从26°c 移至32摄氏度。</li>
		<li>在3或4天以后, 分裂文化1:5 在 amastigote 媒介在32°c。</li>
		<li>检查寄生虫在未来 3-4 天 (最多7天), 看看他们是否可以使用的感染。 注意: 健康的无菌 amastigotes 应该有一个椭圆形的形状, 没有可见的鞭毛。部份地被区分的 amastigotes 有一个大卵形与短鞭毛。这种文化不应该有大量的团簇--许多团簇是不生长的、垂死的寄生虫的征兆。amastigote 文化可以被分裂1:5 和保持至多3星期。</li>
	</ol>
</li>
</ol>4. 感染L. amazonensis
<ol><li>根据所需的语言 (通常为每巨噬细胞 3-5 metacyclic 前鞭毛体 (1:3 或 1:5) 和 1 amastigote 每巨噬细胞 [语言 1:1]) 稀释寄生虫悬浮液。在 PBS 中添加利什曼原虫--在体积为 50-100 µL 的每一个已包含 2 mL 介质的井中。</li>
	<li>用 metacyclic 前鞭毛体34o C BMMs 1 小时的 amastigotes 和3小时孵育, 以进行感染。 注: 最佳感染温度可能因种类而异。</li>
	<li>跟随孵化, 彻底地洗涤在每个井的自由寄生虫3x 与2毫升 PBS-预热到37°c。 注: 轻轻分散 PBS, 确保盖玻片不漂浮在一起。轻轻地旋转盘子, 然后吸出液体。如果使用多种寄生虫, 避免交叉污染, 使用单独的吸气尖端。</li>
	<li>固定1小时或3小时的时间点样品通过孵化每个井与 1.5-2 毫升2% 粉煤灰在 pbs-/-为10分钟洗3x 与 pbs-/。不要在 PBS 中吸入最后的洗涤和冷藏的盘子, 直到染色盖玻片。</li>
	<li>增加2毫升新鲜法钢公司培养基到每个井在板材为进一步时间点和孵化在34°c。</li>
	<li>按照步骤4.3 中的描述, 修正剩余的时间点, 并冷藏盘子直到染色。</li>
</ol>5. DAPI 染色和盖玻片安装
<ol><li>从含有盖玻片的水井中吸出 pbs, 并添加1.5 毫升 pbs--与0.1% 非离子洗涤剂。在室温下孵育10分钟, 然后用2毫升 PBS-/-进行3x 洗涤。</li>
	<li>添加1毫升 PBS-/-与2µg/毫升 DAPI (稀释从5毫克/毫升溶液在水中) 到含盖玻片的水井, 在室温下孵育另1小时。 注意: DAPI 的潜伏期对细胞内利什曼原虫寄生虫的正确可视化至关重要。巨噬细胞细胞核染色迅速, 由于其大小和 DNA 含量较大, 但细胞内寄生虫的细胞核染色较慢。因此, 延长孵化时间超出所需的可视化法钢公司核是必要的, 以允许 DAPI 渗透和寄生虫的等离子膜, 和寄生虫核膜。通过适当的 DAPI 染色, 除了核 DNA 外, 寄生在鞭毛口袋附近的线粒体 kinetoplast dna 也可以被可视化。</li>
	<li>用2毫升 PBS 冲洗每一个含盖玻片3x 的井, 然后用镊子抬起并翻转盖玻片, 将细胞侧向下并安装在玻璃显微镜上, 并提供商用 antifade 安装试剂。 注意: 盖玻片非常脆弱, 需要谨慎处理。避免给他们施加压力, 尤其是在提升时对井侧壁施加压力。添加几滴 PBS--减少了盖玻片和井之间的表面张力, 促进了提升过程。细量规针可用于帮助从井底窥探盖玻片。在幻灯片上放置一个盖玻片后, 轻轻地按下钳子, 以推出安装介质中的气泡。如果盖玻片在起重过程中损坏或有可能翻转, 请勿重新装载;只需使用备用的第四盖玻片。</li>
	<li>将幻灯片冷藏至量化。</li>
</ol>6. 感染量化
<ol><li>在荧光显微镜下检查 DAPI 染色的幻灯片, 方法是使用100X 物镜与浸没油 (358 nm 的激发) 来聚焦巨噬细胞; 最佳排放量为 461 nm。 确保焦点在玻璃滑块和盖玻片之间的巨噬细胞层上。</li>
	<li>量化的巨噬细胞数量 (与 DAPI 染色的大核) 和小的 amastigote 核聚集在每个巨噬细胞核周围 (参见图 2A, DAPI) 为每个视野, 使用手动计数器。 注: Amastigote 核由于体积小, 在一平面上可能不会全部可见。计算那些在巨噬细胞核上可见的, 然后用细焦点检查在巨噬细胞核上方和下方的飞机上的寄生虫核。</li>
	<li>移动到另一个视野, 重复量化。使用单独的计数器键跟踪计数的字段数。在每个盖玻片中通过并行行中的可视字段移动, 以防止计数重叠。</li>
	<li>每盖玻片的最小200只巨噬细胞数量。量化每一次感染的时间点 (3 盖玻片)。如果前一个不适合计数由于盖玻片重叠, 安装不良, 玻璃破损, 等, 请计算第四盖玻片。 注: 如果200只巨噬细胞不能在任何一个盖玻片上计数, 请计算备用的第四。</li>
	<li>计算感染率为 amastigotes/巨噬细胞或 amastigotes/100 巨噬细胞, 并确定从原始量化数据中感染的巨噬细胞百分比。</li>
</ol>

作者声明他们没有竞争的金融利益,

上述法钢公司感染试验所产生的定量数据, 使调查人员能够在相对较短的时间内获得感染率和可靠地测定毒力特性的变化 (最多2周, 比2体内实验所需的月份)。这种方法依赖于 DNA 特异染料 DAPI, 专门染色巨噬细胞和寄生虫细胞核, 并允许快速识别和量化感染细胞。相比之下, 其他污渍, 如姬姆萨绑定到大量不同的细胞分量, 具有不同的强度, 复杂的视觉分析37。使用 DAPI 允许识?...

利什曼病是指由利什曼原虫属的原虫寄生种类引起的广泛的人类疾病。目前约有1200万人感染了全球范围内的利什曼原虫, 3.5亿多名患者面临风险。疾病病理学依赖于利什曼原虫物种和寄主因素, 症状不同于无害的自我愈合皮肤病变和致命的 visceralizing 形式。如果未经治疗, 内脏利什曼病是致命的, 只有在疟疾后才会成为感染了原生动物寄生虫的最致命的人类疾病1。尽管疾病病理和症状存在广泛的差异, 但所有的利什曼原虫物种分别在昆虫和脊椎动物寄主的 promastigote 和 amastigote 阶段之间有 digenic 的生命周期交替。在脊椎动物体内,利什曼原虫目标宿主巨噬细胞侵入, 并诱导 parasitophorous 液泡 (PVs) 的形成, 酸性隔层具有高度剧毒 amastigote 形态复制的 phagolysosomes 特性。Amastigotes 持续在宿主组织中的慢性感染, 可以传递到未感染的白蛉, 完成传输周期。因此, 在人类疾病发展的背景下, amastigotes 是最重要的利什曼原虫生命周期表单2。调查 amastigotes 如何在巨噬细胞 PVs 中复制对于了解利什曼原虫毒力3,4,5,6,7和新的有效疗法的发展。
我们在这里描述了我们的实验室经常使用的方法来研究在骨髓源巨噬细胞 (BMMs) 中的利什曼原虫感染和复制, 这涉及到定量评估细胞内的利什曼原虫的数量随着时间的推移。该过程包括从小鼠骨髓中获取单核细胞和分化为培养的巨噬细胞,体外感染与感染型 (metacyclic 前鞭毛体或 amastigotes)的利什曼原虫和量化的每隔24小时胞内寄生虫的数量在感染后 72-96 小时内。本试验已在实验室中使用, 以确定几种环境因素和寄生虫基因的影响, 包括鉴定铁在促进L. amazonensis毒力方面的关键作用, 进一步验证了都安小鼠病变发育研究6,8,9,10,11,12,13,14,15.由于所有致病性的利什曼原虫物种都在宿主巨噬细胞内建立了它们的复制利基, 这种检测方法可普遍用于所有利什曼原虫物种的毒力测定。
执行法钢公司感染可以分析单个细胞层面上的寄主寄生虫相互作用, 从而更广泛地了解利什曼原虫寄生虫如何与他们的首选宿主微环境、巨噬细胞的 PVs 相互作用。巨噬细胞感染化验已成功使用多个组16,17,18,19,20,21,22 , 探索宿主巨噬细胞和利什曼原虫特定基因的功能, 以及它们在细胞内感染的复杂相互作用中的潜在参与。法钢公司感染允许将寄生虫生长量量化为对影响细胞内生存的宿主因素的影响的读出, 如杀菌剂一氧化氮生产、产生活性氧种类和其他不利条件在溶酶体样 PVs23中遇到。巨噬细胞感染的检测也被用来识别潜在的抗 leishmanial 药物治疗发展的线索13,24。
法钢公司感染的体外性质比其他评估利什曼原虫毒力的方法具有多种优势。然而, 以前的几项研究表明, 随着时间的推移, 细胞内寄生虫存活的机制并没有量化感染率为20,21,24。此外, 许多研究侧重于以下的体内感染随着时间的推移, 通过测量皮肤病变大小和其他生理症状只是间接相关的寄生虫复制25,26,27.体内感染是一种严格的评估寄生虫毒力的方法, 但仅基于都安肿胀的病变大小测量往往是不够的, 因为它们反映了感染组织的炎症反应, 而不是寄生虫的绝对数量。因此, 都安病变的发展化验必须遵循量化的寄生虫负荷的感染组织, 这一程序需要冗长的限制稀释化验28。此外,体内研究通常涉及在不同时间点牺牲多个动物以提取感兴趣的组织6,8,9,10,11,13. 相比之下, 大量的 BMMs 可以从一只动物身上获得, 这些细胞可以在允许在不同时间点对感染进行评估的条件下进行镀膜。此外, 与体内研究相比, 执行体外法钢公司感染可以更好地控制实验条件。量化的巨噬细胞感染与寄生虫本身允许精确控制感染的多样性 (语言) 和文化条件。对这些因素的精细控制是识别离散细胞通路特征和了解它们对感染过程的影响的关键。
考虑到这些优势, 对于研究利什曼原虫毒力的少数群体迄今充分利用细胞内复制的定量评估, 这一点令人吃惊。在本文中, 我们讨论了可能妨碍更广泛地利用此检测的常见缺陷, 并提供了一步一步的协议, 以促进其适当的实施。考虑到它的精确性和通用性, 我们在这里描述的法钢公司感染检测方法不仅可以用来探索影响利什曼原虫毒力的寄主病原体相互作用, 而且还能研究在体内复制的其他微生物。巨噬细胞29。重要的是, 这种检测也可以作为一种快速、经济的临床前筛选方法, 用于抗 leishmanial 药物的开发。

<table><tbody><tr><td>6 well cell culture plate</td><td>Cellstar</td><td>657160</td><td></td></tr><tr><td>Adenine</td><td>Acros Organics</td><td>AC147440250</td><td></td></tr><tr><td>Aerosol Barrier Pipet Tips (100-1000 &amp;mu;L)</td><td>Fisherbrand</td><td>02-707-404</td><td></td></tr><tr><td>Aerosol Barrier Pipet Tips (20-200 &amp;mu;L)</td><td>Fisherbrand</td><td>02-707-430</td><td></td></tr><tr><td>Aerosol Barrier Pipet Tips (2-20 &amp;mu;L)</td><td>Fisherbrand</td><td>02-707-432</td><td></td></tr><tr><td>Bard-Parker Rib-Back Carbon Steel Surgical Blade #10</td><td>Aspen Surgical</td><td>371110</td><td></td></tr><tr><td>BD Luer-Lok Tip 10 mL Syringe</td><td>Becton Dickinson (BD)</td><td>309604</td><td></td></tr><tr><td>BD Precisionglide Needle, 25G&amp;nbsp;</td><td>Becton Dickinson (BD)</td><td>305124</td><td></td></tr><tr><td>Cell Culture Dish 35 mm x 10 mm</td><td>Cellstar</td><td>627 160</td><td></td></tr><tr><td>Cell Culture Flask</td><td>Cellstar</td><td>660175</td><td></td></tr><tr><td>Cover Glasses: 12 mm circles</td><td>Fisherbrand</td><td>12-545-80</td><td></td></tr><tr><td>DAPI (4&#039;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td><td>Invitrogen</td><td>D1306</td><td></td></tr><tr><td>D-Biotin</td><td>J.T. Baker</td><td>C272-00</td><td></td></tr><tr><td>EDTA</td><td>Sigma Aldrich</td><td>EDS</td><td></td></tr><tr><td>Ethyl alcohol 200 proof</td><td>Pharmco-AAPER</td><td>111000200</td><td></td></tr><tr><td>Falcon 100 mm x 15 mm non-TC-treated polystyrene Petri dish</td><td>Corning</td><td>351029</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Seradigm</td><td>1500-500</td><td></td></tr><tr><td>Ficoll400</td><td>Sigma Aldrich</td><td>F8016</td><td></td></tr><tr><td>Fluorescence Microscope</td><td>Nikon</td><td>E200</td><td></td></tr><tr><td>Goat anti-mouse IgG Texas red</td><td>Invitrogen</td><td>T-862</td><td></td></tr><tr><td>Goat anti-rabbit IgG AlexaFluor488</td><td>Invitrogen</td><td>A-11034</td><td></td></tr><tr><td>Hemin</td><td>Tokyo Chemical Industry Co. LTD</td><td>H0008</td><td></td></tr><tr><td>HEPES (1M)</td><td>Gibco</td><td>15630-080</td><td></td></tr><tr><td>Isoton II Diluent</td><td>Beckman Coulter</td><td>8546719</td><td></td></tr><tr><td>L-Glutamine</td><td>Gemini</td><td>400-106</td><td></td></tr><tr><td>Medium 199 (10X)</td><td>Gibco</td><td>11825-015</td><td></td></tr><tr><td>Na pyruvate (100 mM)</td><td>Gibco</td><td>11360-070</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Alfa Aesar</td><td>43368</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Gemini</td><td>400-109</td><td></td></tr><tr><td>Phosphate Buffered Saline (-/-)</td><td>ThermoFisher</td><td>14200166</td><td></td></tr><tr><td>Polypropyline conical Centrifuge Tubes 15 mL</td><td>Cellstar</td><td>188 271</td><td></td></tr><tr><td>Polypropyline conical Centrifuge Tubes 50 mL</td><td>Cellstar</td><td>227 261</td><td></td></tr><tr><td>ProLong Gold antifade reagent</td><td>ThermoFisher</td><td>P36930</td><td></td></tr><tr><td>Rat anti-mouse Lamp-1 antibody</td><td>Developmental Studies Hybridoma Bank</td><td>1D4B</td><td></td></tr><tr><td>Recombinant Human M-CSF</td><td>PeproTech</td><td>300-25</td><td></td></tr><tr><td>Reichert Bright-Line&amp;nbsp; Hemocytometer&amp;nbsp;</td><td>Hausser Scientific</td><td>1492</td><td></td></tr><tr><td>RPMI Medium 1640 (1X)</td><td>Gibco</td><td>11875-093</td><td></td></tr><tr><td>Triton X-100 Surfactant</td><td>Millipore Sigma</td><td>TX1568-1</td><td></td></tr><tr><td>Trypan Blue</td><td>Sigma Aldrich</td><td>T8154</td><td></td></tr><tr><td>Delicate Scissors, 4 1/2&quot;</td><td>VWR</td><td>82027-582</td><td></td></tr><tr><td>Dissecting Forceps, Fine Tip</td><td>VWR</td><td>82027-386</td><td></td></tr><tr><td>Microscope Slides</td><td>VWR</td><td>16004-368</td><td></td></tr><tr><td>Z1 Coulter Particle Counter, Dual Threshold</td><td>Beckman Coulter</td><td>6605699</td><td></td></tr></tbody></table>

quantification of intracellular growth inside macrophages is a fast and reliable method for assessing the virulence of leishmania parasites

利什曼原虫的生命周期, 利什曼病的致病剂, 分别在昆虫和脊椎动物寄主 promastigote 和 amastigote 阶段交替。虽然利什曼病的致病症状可能有很大差异, 从良性皮肤病变到高度致命的内脏疾病的形式取决于感染的物种, 所有的利什曼原虫物种驻留在宿主巨噬细胞在脊椎动物阶段他们的生命周期。因此,利什曼原虫传染性直接关系到它在巨噬细胞内 parasitophorous 泡 (PVs) 中侵入、生存和复制的能力。因此, 评估寄生虫复制 intracellularly 的能力是确定毒力的可靠方法。研究利什曼病的发展使用动物模型是费时, 繁琐, 往往是困难的, 特别是与 pathogenically 重要的内脏形式。我们这里描述了一个方法来遵循的细胞内发育的利什曼原虫骨髓源性巨噬细胞 (BMMs)。细胞内寄生虫的数量是确定的24小时间隔为 72-96 h 继感染后。这种方法可以可靠地确定各种遗传因素对利什曼原虫毒力的影响。举例来说, 我们展示了单个等位基因删除利什曼原虫线粒体铁转运基因 (LMIT1) 如何削弱利什曼原虫 amazonensis突变株LMIT1/ΔLmit1在 BMMs 内生长的能力,反映了与野生型相比, 毒力的急剧减少。这种检测还允许精确控制实验条件, 可以单独操作, 分析各种因素 (营养素、活性氧种类、等等) 对寄主-病原体相互作用的影响。因此, 适当的执行和量化的法钢公司感染研究提供了一个非侵入性, 快速, 经济, 安全和可靠的替代传统动物模型研究。

利什曼原虫有两种受感染的窗体-metacyclic 前鞭毛体, 它们区别于区域性的固定阶段的 procyclic 前鞭毛体, amastigotes 是胞内阶段(图 1)。在某些利什曼原虫种 (如L. amazonensis) 中, amastigotes 也可以通过将 promastigote 细胞转移到较低的 pH 值 (4.5) 和高温 (32 °c), 与法钢公司中发现的条件类似, 在无菌文化中进行区分 PVs8,</s...

Watch this Scientific Journal Video about Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites at JoVE.com

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

所有致病性的利什曼原虫物种在其脊椎动物宿主的巨噬细胞内栖息和复制。在这里, 我们提出了一个协议, 以感染小鼠骨髓源性巨噬细胞的文化与利什曼原虫, 然后精确定量的细胞内生长动力学。该方法对于研究影响寄主病原体相互作用和利什曼原虫毒力的个体因素非常有用。

巨噬细胞内细胞内生长的定量化是评价利什曼原虫寄生虫毒力的快速可靠方法

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate ...

quantification-intracellular-growth-inside-macrophages-is-fast

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

10.4K 次观看.  University of Maryland. 所有致病性的利什曼原虫物种在其脊椎动物宿主的巨噬细胞内栖息和复制。在这里, 我们提出了一个协议, 以感染小鼠骨髓源性巨噬细胞的文化与利什曼原虫, 然后精确定量的细胞内生长动力学。该方法对于研究影响寄主病原体相互作用和利什曼原虫毒力的个体因素非常有用。

视频: Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

在Live主机转利什曼原虫寄生虫在体内成像

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

科研

免疫与感染

Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to successful identification of genes or loci underlying various traits in malaria parasites of rodents1-3 and humans4-6. The malaria parasite Plasmodium yoelii is one of many malaria species isolated from wild African rodents and has been adapted to grow in laboratories. This species reproduces many of the biologic characteristics of the human malaria parasites; genetic markers such as microsatellite and amplified fragment length polymorphism (AFLP) markers have also been developed for the parasite7-9. Thus, genetic studies in rodent malaria parasites can be performed to complement research on Plasmodium falciparum. Here, we demonstrate the techniques for producing a genetic cross in P. yoelii that were first pioneered by Drs. David Walliker, Richard Carter, and colleagues at the University of Edinburgh10.

Genetic crosses in P. yoelii and other rodent malaria parasites are conducted by infecting mice Mus musculus with an inoculum containing gametocytes of two genetically distinct clones that differ in phenotypes of interest and by allowing mosquitoes to feed on the infected mice 4 days after infection. The presence of male and female gametocytes in the mouse blood is microscopically confirmed before feeding. Within 48 hrs after feeding, in the midgut of the mosquito, the haploid gametocytes differentiate into male and female gametes, fertilize, and form a diploid zygote (Fig. 1). During development of a zygote into an ookinete, meiosis appears to occur11. If the zygote is derived through cross-fertilization between gametes of the two genetically distinct parasites, genetic exchanges (chromosomal reassortment and cross-overs between the non-sister chromatids of a pair of homologous chromosomes; Fig. 2) may occur, resulting in recombination of genetic material at homologous loci. Each zygote undergoes two successive nuclear divisions, leading to four haploid nuclei. An ookinete further develops into an oocyst. Once the oocyst matures, thousands of sporozoites (the progeny of the cross) are formed and released into mosquito hemoceal. Sporozoites are harvested from the salivary glands and injected into a new murine host, where pre-erythrocytic and erythrocytic stage development takes place. Erythrocytic forms are cloned and classified with regard to the characters distinguishing the parental lines prior to genetic linkage mapping. Control infections of individual parental clones are performed in the same way as the production of a genetic cross.

Genetic crosses of rodent malaria parasites are performed by feeding two genetically distinct parasites to mosquitoes. Recombinant progeny are cloned from mouse blood after allowing mosquitoes to bite infected mice. This video shows how to produce genetic crosses of Plasmodium yoelii and is applicable to other rodent malaria parasites.

生产的灭鼠疟疾寄生虫的遗传交叉议定书“

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to ...

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

Leishmaniasis is one of the world's most neglected diseases, largely affecting the poorest of the poor, mainly in developing countries. Over 350 million people are considered at risk of contracting leishmaniasis, and approximately 2 million new cases occur yearly1. Leishmania donovani is the causative agent for visceral leishmaniasis (VL), the most fatal form of the disease. The choice of drugs available to treat leishmaniasis is limited 2;current treatments provide limited efficacy and many are toxic at therapeutic doses. In addition, most of the first line treatment drugs have already lost their utility due to increasing multiple drug resistance 3. The current pipeline of anti-leishmanial drugs is also severely depleted. Sustained efforts are needed to enrich a new anti-leishmanial drug discovery pipeline, and this endeavor relies on the availability of suitable in vitro screening models.
In vitro promastigotes 4 and axenic amastigotes assays5 are primarily used for anti-leishmanial drug screening however, may not be appropriate due to significant cellular, physiological, biochemical and molecular differences in comparison to intracellular amastigotes. Assays with macrophage-amastigotes models are considered closest to the pathophysiological conditions of leishmaniasis, and are therefore the most appropriate for in vitro screening. Differentiated, non-dividing human acute monocytic leukemia cells (THP1) (make an attractive) alternative to isolated primary macrophages and can be used for assaying anti-leishmanial activity of different compounds against intracellular amastigotes.
Here, we present a parasite-rescue and transformation assay with differentiated THP1 cells infected in vitro with Leishmania donovani for screening pure compounds and natural products extracts and determining the efficacy against the intracellular Leishmania amastigotes. The assay involves the following steps: (1) differentiation of THP1 cells to non-dividing macrophages, (2) infection of macrophages with L. donovani metacyclic promastigotes, (3) treatment of infected cells with test drugs, (4) controlled lysis of infected macrophages, (5) release/rescue of amastigotes and (6) transformation of live amastigotes to promastigotes. The assay was optimized using detergent treatment for controlled lysis of Leishmania-infected THP1 cells to achieve almost complete rescue of viable intracellular amastigotes with minimal effect on their ability to transform to promastigotes. Different macrophage:promastigotes ratios were tested to achieve maximum infection. Quantification of the infection was performed through transformation of live, rescued Leishmania amastigotes to promastigotes and evaluation of their growth by an alamarBlue fluorometric assay in 96-well microplates. This assay is comparable to the currently-used microscopic, transgenic reporter gene and digital-image analysis assays. This assay is robust and measures only the live intracellular amastigotes compared to reporter gene and image analysis assays, which may not differentiate between live and dead amastigotes. Also, the assay has been validated with a current panel of anti-leishmanial drugs and has been successfully applied to large-scale screening of pure compounds and a library of natural products fractions (Tekwani et al. unpublished).

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

A parasite-rescue and transformation assay with THP1 cells infected in vitro with Leishmania donovani has been optimized for anti-leishmanial drug screening. The assay involves differentiation of THP1 cells, infection with promastigotes, treatment with test drugs, controlled lysis of the infected macrophages, rescue of amastigotes, transformation to promastigotes and monitoring promastigote growth and proliferation with a fluorometric assay.

Antileishmanial筛选对细胞内的寄生虫救援和转化实验<em&gt;杜氏利什曼原虫</em无鞭毛体在THP1人急性单核细胞白血病细胞株

Leishmaniasis is  one of the world's most neglected diseases, largely affecting the  poorest of the poor, mainly in developing countries. Over 350 million ...

In Vivo Infection with Leishmania amazonensis to Evaluate Parasite Virulence in Mice

Leishmania spp. are protozoan parasites that cause leishmaniases, diseases that present a wide spectrum of clinical manifestations from cutaneous to visceral lesions. Currently, 12 million people are estimated to be infected with Leishmania worldwide and over 1 billion people live at the risk of infection. Leishmania amazonensis is endemic in Central and South America and usually leads to the cutaneous form of the disease, which can be directly visualized in an animal model. Therefore, L. amazonensis strains are good models for cutaneous leishmaniasis studies because they are also easily cultivated in vitro. C57BL/6 mice mimic the L. amazonensis-driven disease progression observed in humans and are considered one of the best mice strains model for cutaneous leishmaniasis. In the vertebrate host, these parasites inhabit macrophages despite the defense mechanisms of these cells. Several studies use in vitro macrophage infection assays to evaluate the parasite infectivity under different conditions. However, the in vitro approach is limited to an isolated cell system that disregards the organism&#39;s response. Here, we compile an in vivo murine infection method that provides a systemic physiological overview of the host-parasite interaction. The detailed protocol for the in vivo infection of C57BL/6 mice with L. amazonensis comprises parasite differentiation into infective amastigotes, mice footpad cutaneous inoculation, lesion development, and parasite load determination. We propose this well-established method as the most adequate method for physiological studies of the host immune and metabolic responses to cutaneous leishmaniasis.

In Vivo Infection with Leishmania amazonensis to Evaluate Parasite Virulence in Mice

Here, we present a compiled protocol to evaluate the cutaneous infection of mice with Leishmania amazonensis. This is a reliable method for studying parasite virulence, allowing a systemic view of the vertebrate host response to the infection.

在 Vivo 感染利什曼病亚马孙病评估小鼠的寄生虫毒性

Leishmania spp. are protozoan parasites that cause leishmaniases, diseases that present a wide spectrum of clinical manifestations from cutaneous to ...

Investigating the Phagocytosis of Leishmania using Confocal Microscopy

Phagocytosis is an orchestrated process that involves distinct steps: recognition, binding, and internalization. Professional phagocytes take up Leishmania parasites by phagocytosis, consisting of recognizing ligands on parasite surfaces by multiple host cell receptors. Binding of Leishmania to macrophage membranes occurs through complement receptor type 1 (CR1) and complement receptor type 3 (CR3) and Pattern Recognition Receptors. Lipophosphoglycan (LPG) and 63 kDa glycoprotein (gp63) are the main ligands involved in macrophage-Leishmania interactions. Following the initial recognition of parasite ligands by host cell receptors, parasites become internalized, survive, and multiply within parasitophorous vacuoles. The maturation process of Leishmania-induced vacuoles involves the acquisition of molecules from intracellular vesicles, including monomeric G protein Rab 5 and Rab 7, lysosomal associated membrane protein 1 (LAMP-1), lysosomal associated membrane protein 2 (LAMP-2), and microtubule-associated protein 1A/1B-light chain 3 (LC3).
Here, we describe methods to evaluate the early events occurring during Leishmania interaction with the host cells using confocal microscopy, including (i) binding (ii) internalization, and (iii) phagosome maturation. By adding to the body of knowledge surrounding these determinants of infection outcome, we hope to improve the understanding of the pathogenesis of Leishmania infection and support the eventual search for novel chemotherapeutic targets.

Investigating the Phagocytosis of Leishmania using Confocal Microscopy

The mechanism associated with phagocytosis in Leishmania infection remains poorly understood. Here, we describe methods to evaluate the early events occurring during Leishmania interaction with the host cells.

使用共聚焦显微镜研究 利什曼原虫 的吞噬作用

Phagocytosis is an orchestrated process that involves distinct steps: recognition, binding, and internalization. Professional phagocytes take up ...

Obtainment of Macrophages from Human Monocytes to Assess Leishmania braziliensis Infection Rate and Innate Host Immune Response

Macrophages are multifunctional cells essential to the immune system function, and the primary host cell in Leishmania braziliensis (Lb) infection. These cells are specialized in microorganism recognition and phagocytosis, but also activate other immune cells and present antigens, as well as promote inflammation and tissue repair. Here, we describe a protocol to obtain mononuclear cells from peripheral blood (PBMC) of healthy donors to separate monocytes that then differentiate into macrophages. These cells can then be infected in vitro at different Lb concentrations to evaluate the ability to control infection, as well as evaluate host cell immune response, which can be measured by several methods. PBMCs were first isolated by centrifuging with Ficoll-Hypaque gradient and then plated to allow monocytes to adhere to culture plates; non-adherent cells were removed by washing. Next, adherent cells were cultured with macrophage-colony stimulating factor (M-CSF) for 7 days to induce macrophage differentiation. We suggest plating 2 x 106&#160;cells per well on 24-well plates in order to obtain 2 x 105&#160;macrophages. Fully differentiated macrophages can then be infected with Lb for 4 or 24 hours. This protocol results in a significant percentage of infected cells, which can be assessed by optical or fluorescence microscopy. In addition to infection index, parasite load can be measured by counting the numbers of parasites inside each cell. Further molecular and functional assays can also be performed in culture supernatants or within the macrophages themselves, which allows this protocol to be applied in a variety of contexts and also adapted to other intracellular parasite species.

Obtainment of Macrophages from Human Monocytes to Assess Leishmania braziliensis Infection Rate and Innate Host Immune Response

This protocol describes the process for obtaining human macrophages from monocytes for infection with Leishmania braziliensis. It also allows researchers to evaluate infection rate and parasite viability, ROS production by fluorescence microscopy, and the production of inflammatory mediators in culture supernatants to investigate macrophage response to infection.

Macrophages are multifunctional cells essential to the immune system function, and the primary host cell in Leishmania braziliensis (Lb) infection. These ...

In Vitro Differentiation of Human Dendritic Cells and their Markers in Leishmania Infection

Leishmaniasis comprises a collection of clinical manifestations associated with the infection of obligate intracellular protozoans, Leishmania. The life cycle of Leishmania parasites consists of two alternating life stages (amastigotes and promastigotes), during which parasites reside within either arthropod vectors or vertebrate hosts, respectively. Notably, the complex interactions between Leishmania parasites and several cells of the immune system largely influence the outcome of infection. Importantly, although macrophages are known to be the main host niche for Leishmania replication, parasites are also phagocytosed by other innate immune cells, such as neutrophils and dendritic cells (DCs). 
DCs play a major role in bridging the innate and adaptive branches of immunity and thus orchestrate immune responses against a wide range of pathogens. The mechanisms by which Leishmania and DCs interact remain unclear and involve aspects of pathogen capture, the dynamics of DC maturation and activation, DC migration to draining lymph node (dLNs), and antigen presentation to T cells. Although a large body of studies support the notion that DCs play a dual role in modulating immune responses against Leishmania, the participation of these cells in susceptibility or resistance to Leishmania remains poorly understood. After infection, DCs undergo a maturation process associated with the upregulation of surface major histocompatibility complex (MHC) II, in addition to costimulatory molecules (namely, CD40, CD80, and CD86). 
Understanding the role of DCs in infection outcome is crucial to developing therapeutic and prophylactic strategies to modulate the immune response against Leishmania. This paper describes a method for the characterization of Leishmania-DC interaction. This detailed protocol provides guidance throughout the steps of DC differentiation, the characterization of cell surface molecules, and infection protocols, allowing scientists to investigate DC response to Leishmania infection and gain insight into the roles played by these cells in the course of infection.

In Vitro Differentiation of Human Dendritic Cells and their Markers in Leishmania Infection

Dendritic cells (DCs) are essential components of innate immunity against Leishmania infection. The mechanisms underlying the complex interaction between DCs and Leishmania remain poorly understood. Here, we describe methods to evaluate how Leishmania infection affects the immunobiological function of human DCs, such as migration-related and costimulatory molecule expression.

Leishmaniasis comprises a collection of clinical manifestations associated with the infection of obligate intracellular protozoans, Leishmania. The life ...

In Vivo Imaging of Leishmania infantum-infected Hamsters by Gingival Inoculation of Axenic Amastigotes Expressing Luciferase

American tegumentary leishmaniasis (ATL) and visceral leishmaniasis (VL) are considered neglected by the World Health Organization. VL can be lethal if not treated; the drugs used in treatment are toxic, and there are cases of resistance. Preclinical tests can represent a bottleneck in discovering new medicines for treatment, depending on the animal model, the strain used, and the inoculum route. The golden hamster stands out for its high susceptibility to subgenera Viannia and Leishmania species, displaying many of the clinical and immunopathological processes observed in human disease.
By hamster anatomy, which has a short tail and limbs, the intracardiac route is usually the choice for intravenous injection of Leishmania. However, it is an inoculum that can lead to bleeding and eventually to animal death. Thus, we standardized an alternative intravenous inoculation route for infection at the gingival vein, which is minimally invasive, allows easy venous access, and causes few local and systemic injuries to the animal. Therefore, hamsters infected by the intraperitoneal (IP) or intragingival (IG) route with Leishmania Infantum expressing luciferase (Luc) were followed up for 22 days by the bioluminescence imaging system and 50 days and 8 months post infection by PCR.
After gingival inoculation of both axenic amastigotes and promastigotes of L. infantum-Luc, bioluminescence was restricted for at least 2 weeks at the site of injection, which is an indicator of infection in the tissues around the gingival plexus. Hamsters infected intraperitoneally with L. infantum-Luc displayed bioluminescence dispersed throughout the abdomen, as expected. However, by the bioluminescence imaging system infection declined until the 50th dpi and was only detectable by PCR.&#160;Axenic amastigotes showed better infection than promastigotes, as evaluated by PCR. Indeed, 8 months after infection, parasites were detected by PCR in the liver of animals inoculated with axenic amastigotes by the intravenous route, which can be a characteristic of the reference strain of L. infantum MHOM/BR/1974/PP75, whose infection progresses slowly and display low parasite burden, below the bioluminescent imaging resolution. Thus, axenic amastigotes can be a better choice for infection and follow-up than promastigotes, and the gingival inoculum is a feasible route for intravenous injection of Leishmania and other pathogens.

Here we present a longitudinal evaluation of golden hamsters infected intraperitoneally (IP) or via intragingival (IG) with L. infantum-Luc by bioluminescent imaging and by PCR. Hamsters were evaluated 1 day post infection (1 dpi), 1 week after infection (8 dpi), and 3 weeks after infection (22 dpi) and euthanized at the 50th dpi and 8 months post infection.

体内研究 通过牙龈接种表达荧光素酶的轴丝无毛体对 婴儿利什曼原虫感染的仓鼠进行成像

American tegumentary leishmaniasis (ATL) and visceral leishmaniasis (VL) are considered neglected by the World Health Organization. VL can be lethal if ...

生物学

Assessment of Leishmania Virulence within Cultured Macrophages

Source: Sarkar, A. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/57486">Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of&#160;Leishmania&#160;Parasites.</a>&#160;J. Vis. Exp.&#160;(2018)
This video investigates Leishmania&#39;s virulence by treating macrophages with metacyclic promastigotes, resulting in the formation of intracellular amastigotes. The confirmation of Leishmania&#39;s virulence is achieved through nucleic acid fluorescent dye staining. This insight can contribute to the development of new therapies for leishmaniasis.

培养巨噬细胞中利什曼原虫毒力的评估

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Imaging and Visualization Techniques

An In Vitro Assay to Quantify the Intracellular Growth of Parasite in Macrophages

Source: Sarkar, A., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/57486">Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites.</a> J. Vis. Exp. (2018).
This video demonstrates an assay to quantify the intracellular growth of parasites in bone marrow-derived macrophages (BMMs). The macrophage cells are infected with the motile and non-motile invasive stages of Leishmania, metacyclic promastigotes and amastigotes, respectively. The parasites invade the macrophages, getting internalized into a parasitophorous vacuole and multiplying inside. The intracellular parasite growth is quantified using a microscope upon fluorescently staining the macrophage and parasite nuclei.

一种定量巨噬细胞中寄生虫细胞内生长的体外测定

1. Plating BMMs on Coverslips for Infection 

	Prepare 6-well tissue culture plates by placing four (4) sterile 12 mm glass coverslips (autoclaved and ...

巨噬细胞内细胞内生长的定量化是评价利什曼原虫寄生虫毒力的快速可靠方法

摘要

探索更多视频

此视频中的章节

在Live主机转利什曼原虫寄生虫在体内成像

生产的灭鼠疟疾寄生虫的遗传交叉议定书“

Antileishmanial筛选对细胞内的寄生虫救援和转化实验

在 Vivo 感染利什曼病亚马孙病评估小鼠的寄生虫毒性

使用共聚焦显微镜研究利什曼原虫的吞噬作用

Obtainment of Macrophages from Human Monocytes to Assess Leishmania braziliensis Infection Rate and Innate Host Immune Response

In Vitro Differentiation of Human Dendritic Cells and their Markers in Leishmania Infection

体内研究通过牙龈接种表达荧光素酶的轴丝无毛体对婴儿利什曼原虫感染的仓鼠进行成像

培养巨噬细胞中利什曼原虫毒力的评估

一种定量巨噬细胞中寄生虫细胞内生长的体外测定

巨噬细胞内细胞内生长的定量化是评价利什曼原虫寄生虫毒力的快速可靠方法

摘要

探索更多视频

此视频中的章节

在Live主机转利什曼原虫寄生虫在体内成像

生产的灭鼠疟疾寄生虫的遗传交叉议定书“

Antileishmanial筛选对细胞内的寄生虫救援和转化实验

在 Vivo 感染利什曼病亚马孙病评估小鼠的寄生虫毒性

使用共聚焦显微镜研究 利什曼原虫 的吞噬作用

Obtainment of Macrophages from Human Monocytes to Assess Leishmania braziliensis Infection Rate and Innate Host Immune Response

In Vitro Differentiation of Human Dendritic Cells and their Markers in Leishmania Infection

体内研究 通过牙龈接种表达荧光素酶的轴丝无毛体对 婴儿利什曼原虫感染的仓鼠进行成像

培养巨噬细胞中利什曼原虫毒力的评估

一种定量巨噬细胞中寄生虫细胞内生长的体外测定

使用共聚焦显微镜研究利什曼原虫的吞噬作用

体内研究通过牙龈接种表达荧光素酶的轴丝无毛体对婴儿利什曼原虫感染的仓鼠进行成像