Name: use of image cytometry for quantification of pathogenic fungi in association with host cells
Uploaded: 2013-06-19T12:00:00
Description: Watch this Scientific Journal Video about Use of Image Cytometry for Quantification of Pathogenic Fungi in Association with Host Cells at JoVE.com

1。感染的巨噬细胞与H.荚膜，白色念珠菌 <ol><li>在感染前16小时，在所需的密度在24孔板中的种子的巨噬细胞。在这个协议中，使用密度为3.0×10 5个细胞/孔。 </li><li>添加对数期生长的真菌细胞中，在所需的感染复数（MOI）。这个协议可以容纳一系列的MOI（0.2 - 5.0）。对于感染的巨噬细胞与H.荚膜 ，我们使用了MOI为0.2。 </li><li> 1.5小时，让细胞吞噬后，洗巨噬细胞用PBS三次?...

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图像流式细胞仪允许用户捕捉到高质量的图像，并使用专门的软件，进行快速定量细胞。一个潜在的挑战，通过图像分析微生物的发病机制中的字段是一个混合的人口中存在的细胞，包括哺乳动物宿主细胞，微生物计数。在这里，我们表明，图像分析，可用于体外巨噬细胞的感染过程中的量化可行的致病性酵母菌。我们的方法不仅忠实地概括在体外巨噬细胞感染实验期间观察?...

超过一个时间过程或不同的感染条件下，在与主机和/或宿主细胞的致病性真菌的研究经常需要量化的活的真菌细胞。枚举的菌落形成单位（CFU）是活的真菌细胞的数目已被测量的标准方法，通过它，但是，这种技术具有一些缺点和限制。首先，许多真菌物种生长缓慢。可见固体培养基上菌落增长需要1-2周，研究的步伐显着放缓。二，操纵在CFU电镀过程中的样品是一个费力的过程中，由于必须镀几个稀释度，以确保可数的菌落数。第三，CFU的数目通常低于镀活的微生物的数量，因为电镀效率低于100％。例如，二相性真菌病原体荚膜组织胞浆菌的电镀效率可高达90％，但常规低至30％，甚至更低（10％），为相关的真菌双晶片Paracoccidioides巴西 1，2。 白色念珠菌的电镀效率也受到变异3。最后，CFU分析占活和细胞分裂的活性，能够建立在固体培养基上的生长，而在许多情况下，这将是有用的，以确定是否存在和浓度的的死和/或代谢的非活动单元。 以前，一些致病性真菌物种的定量的流式细胞仪的方法已经描述4-6。然而，由于参与共享流式细胞仪用生物安全2级（BSL2）或BSL3级病原体生物安全遏制问题，采用这项技术已经有限。像流式细胞仪，图像仪是一个敏感和快速的细胞定量分析的方法。然而，可以进行图像分析，在一小部分的成本，而比较的结果7 -11。在这里，我们描述了进行图像分析致病真菌与宿主细胞的方法。我们证明了我们的方法使用两个人类病原真菌： 组织胞浆菌和白色念珠菌。H.荚膜是一种二态真菌病原体引起呼吸系统疾病，它生长在人类作为芽殖酵母和肺泡巨噬细胞内复制。 白色念珠菌是人类共生的物种，偶尔会导致念珠菌。我们表明，图像仪可快速定量的可视化能力，加上这些酵母菌。

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use of image cytometry for quantification of pathogenic fungi in association with host cells

如白色念珠菌 ， 荚膜组织胞浆菌 ， 隐球菌致病酵母细胞发病机制的研究通常采用感染的哺乳动物宿主或宿主细胞（ 如巨噬细胞），然后由酵母定量用集落形成单元分析或流式细胞仪。虽然菌落形成单位枚举在该领域一直是最常用的方法，这种技术的缺点和局限性，包括在固体培养基上，低和/或可变电镀效率，这是比增长时，特别关注一些真菌物种增长缓慢野生型和突变株。流式细胞仪可以提供快速定量有关酵母活力，然而，采用流式细胞仪检测致病性酵母菌已为一些现实的原因，包括其成本高和生物安全方面的考虑限制。在这里，我们展示了一个基于图像使用流式细胞仪的方法Cellometer视觉（Nexcelom生物科技有限责任公司）与巨噬细胞共培养的量化可行的致病性酵母菌。我们的研究集中在两个人的真菌病原体检测： 组织胞浆菌和白色念珠菌。H.荚膜殖民肺泡巨噬细胞通过复制内巨噬细胞的吞噬体，在这里，我们定量评估H.增长荚膜酵母在RAW 264.7巨噬细胞用吖啶橙/碘化丙啶染色结合图像分析。我们的方法忠实地概括增长趋势作为衡量传统的菌落形成单位枚举，但显着增加灵敏度。此外，我们直接评估感染活的巨噬细胞与GFP表达菌株C.白色念珠菌 。我们的方法提供了一种快速，准确和经济的手段，重要关联机智的人类病原真菌的检测和定量h主机细胞。

我们使用Cellometer视觉图像流式细胞仪监测H.增长在巨噬细胞中的荚膜 。 RAW 264.7细胞感染H.荚膜酵母细胞和在不同时间点的样品进行AO / PI染色，随后通过基于图像的流式细胞仪分析。平行样品进行了分析由传统的CFU枚举。在每个时间点，在水中孵育样品裂解巨噬细胞，Cellometer的Vision软件（ 图1a和1b）由解放酵母细胞进行鉴定。我们观察到高度比较可行?...

Watch this Scientific Journal Video about Use of Image Cytometry for Quantification of Pathogenic Fungi in Association with Host Cells at JoVE.com

Use of Image Cytometry for Quantification of Pathogenic Fungi in Association with Host Cells

在这里，我们将演示如何图像流式细胞仪可用于量化致病真菌与宿主细胞培养。 CFU枚举作为一种替代，可以使用这种技术。

使用的细胞图像分析仪量化致病真菌与宿主细胞协会

Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans ...

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Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

Use of the EpiAirway Model for Characterizing Long-term Host-pathogen Interactions

使用的EpiAirway模型，用于描述长期宿主 - 病原体相互作用

Nontypeable Haemophilus influenzae  (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory ...

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

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Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols ...

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

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基于图像的流式细胞仪技术来评估变化粒细胞功能<em&gt;在体外</em

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte ...

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牙龈卟啉单作为模式生物的评价厌氧细菌的相互作用与宿主细胞

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and ...

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During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to ...

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells

基于多色流式细胞仪的 t 细胞线粒体和溶酶体的定量

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular ...

An Advanced Murine Model for Nonalcoholic Steatohepatitis in Association with Type 2 Diabetes

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Obesity is associated with chronic low-grade inflammation and insulin resistance, contributing to an increasing prevalence of chronic metabolic diseases, ...

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

全骨髓的制备,用于中性粒系细胞的大规模细胞测定分析

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...