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三次删除外真菌。 </li><li>孵育前试样分析所需的小时数。受感染的巨噬细胞在低MOI 0.2在我们的实验中，继续生存了好几天，可分析的样品约每12-24小时。 </li></ol> 2。巨噬细胞裂解<ol><li>要解放巨噬细胞的真菌，去除媒体，用PBS洗3次，并添加0.5毫升无菌水。在这些条件下，巨噬细胞溶解和真菌细胞将保持不变。 </li><li>在室温下孵育5分钟。 </li><li>转移裂解无菌试管中，保持在冰上。 </li><li> 20微升裂解液转移到一个单独的管中，再加入20μLAO / PI解决方案。直接进入第5步：“图像流式细胞仪分析样品制备” </li></ol> 3。电镀CFU <ol><li>裂解进行稀释10倍（从2.3步）媒体。 </li><li>铠甲100微升每个稀释度，一式两份，HMM-琼脂糖板。 7-8天，在湿盒中，于37℃5％CO 2孵育板。 </li><li>手动菌落计数平板上显示最低为100，最高1,000个不同的殖民地。 </li></ol> 4。在现场巨噬细胞内真菌的可视化<ol><li>为了收集活的巨噬细胞，洗涤细胞用PBS 3次。加入0.5毫升PBS孵育30分钟，在4°C。</li><li>要除去巨噬细胞组织培养井，轻轻吸管向上和向下几次。液体转移到无菌试管中，保持在冰上。 </li><li> 20微升样品转移到一个单独的试管中，然后加入20μLAO / PI解决方案。直接进入第5步：“图像流式细胞仪分析样品制备”。 </li></ol> 5。图像流式细胞仪分析样品制备<ol><li>彻底移液器的目标样本，然后转移到一次性细胞计数室20微升样品。 </li><li>允许细胞定居室，持续30秒。 </li><li>插入到图像流式细胞仪计数室。 </li></ol> 6。 Cellometer仪器设置<ol><li>荧光光学模块：VB-535-402和VB-660-502插入到系统中，并确保他们锁定到位。 <ol><li> VB-535-402（在475 nm处激发，在535nm处的发射）的是，用于吖啶橙和GFP检测。 </li><li> VB-660-502（excitati在540 nm处，在660nm处的发射）用碘化丙啶检测。 </li></ol></li><li>打开图像流式细胞仪上，并打开附带的软件。 </li></ol> 7。 Cellometer软件设置<ol><li>选择预设“检测类型”和“细胞类型”，在下拉菜单含量。 <ol><li>活力，优化检测吖啶橙和碘化丙啶检测。 </li><li>对于白色念珠菌感染的检测，检测GFP的检测进行了优化。 </li></ol></li><li>在顶部选择“选项”，然后单击“采取背景图片”，并允许操作完成。 </li><li>点击“预览明场图像”。 </li></ol> 8。图像采集程序<ol><li>将图像流式细胞仪的样品室。 </li><li>使用对焦旋钮，调整的重点。 </li><li>一旦聚焦，点击“计数”，并允许图像采集操作完成。 </li><li>圣雷莫VE一次性计数室，并适当处置。 </li></ol> 9。图像数据分析<ol><li>浓度和活力测量<ol><li>一旦完成计数，靶细胞的浓度和生存能力在将显示在结果页面。 </li><li>点击“导出”数据导出到FCS Express 4的白色念珠菌感染实验中的GFP的细胞群体分析。 </li></ol></li><li> 白色念珠菌感染的细胞FCS快速分析<ol><li>导入“。NXDAT”文件复制到FCS Express 4的积荧光直方图的结果。 </li><li>将细胞种群门的直方图来确定白色念珠菌感染的细胞的人口百分比。 </li></ol></li></ol>

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	<li>Worsham, P. L., Goldman, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+plating+of+Histoplasma+capsulatum+without+addition+of+conditioned+medium+or+siderophores.">Quantitative plating of Histoplasma capsulatum without addition of conditioned medium or siderophores.</a> J. Med. Vet. Mycol. 26, 137-143 (1988).</li><li>Goihman-Yahr, M., Pine, L., Albornoz, M. C., Yarzabal, L., de Gomez, M. H., San Martin, B., Ocanto, A., Molina, T., Convit, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+plating+efficiency+and+estimation+of+viability+of+suspensions+of+Paracoccidioides+brasiliensis+yeast+cells.">Studies on plating efficiency and estimation of viability of suspensions of Paracoccidioides brasiliensis yeast cells.</a> Mycopathologia. 71, 73-83 (1980).</li><li>Bhatti, M. 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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+susceptibility+testing+of+Candida+albicans+by+flow+cytometry.">Rapid susceptibility testing of Candida albicans by flow cytometry.</a> Journal of Clinical Microbiology. 35, 358-363 (1997).</li><li>Chan, L. L. -. Y., Lai, N., Wang, E., Smith, T., Yang, X., Lin, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+detection+method+for+apoptosis+and+necrosis+measurement+using+the+Cellometer+imaging+cytometry.">A rapid detection method for apoptosis and necrosis measurement using the Cellometer imaging cytometry.</a> Apoptosis. 16, 1295-1303 (2011).</li><li>Chan, L. L. -. Y., Shen, D., Wilkinson, A. 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作者利奥陈俪颖和本杰明·帕拉迪斯的Nexcelom生物科技有限责任公司的员工。

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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		 <tr>
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			<td>REAGENTS</td>
		 </tr>
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			<td>DMEM</td>
			<td>Life Technologies</td>
			<td>11965-084</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Fetal Bovine Serum</td>
			<td>Life Technologies</td>
			<td>16000044</td>
			<td></td>
		 </tr>
		 <tr>
			<td>AO/PI Solution</td>
			<td>Nexcelom Bioscience</td>
			<td>CSK-0102</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Disposable Counting Chamber</td>
			<td>Nexcelom Bioscience</td>
			<td>CHT4-SD100 </td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>EQUIPMENT</td>
		 </tr>
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			<td>Cellometer Vision</td>
			<td>Nexcelom Bioscience</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Cellometer Vision Software</td>
			<td>Nexcelom Bioscience</td>
			<td></td>
			<td></td>
		 </tr>
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 </table>

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 ...

use-image-cytometry-for-quantification-pathogenic-fungi-association

Research

JoVE Journal

Immunology and Infection

12.8K 次观看.  Merrimack College. 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。

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

Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry:  a Novel Use for a Cell Purification Method

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true quantitative analysis of cellular inflammation. Flow cytometry is a quick alternative method to quantify immune cells in the injured brain or spinal cord tissue. Historically, flow cytometry has been used to quantify immune cells collected from blood or dissociated spleen or thymus, and only a few studies have attempted to quantify immune cells in the injured spinal cord by flow cytometry using fresh dissociated cord tissue. However, the dissociated spinal cord tissue is concentrated with myelin debris that can be mistaken for cells and reduce cell count reliability obtained by the flow cytometer. We have advanced a cell preparation method using the OptiPrep gradient system to effectively separate lipid/myelin debris from cells, providing sensitive and reliable quantifications of cellular inflammation in the injured spinal cord by flow cytometry. As described in our recent study (Beck &amp; Nguyen et al., Brain. 2010 Feb; 133 (Pt 2): 433-47), the OptiPrep cell preparation had increased sensitivity to detect cellular inflammation in the injured spinal cord, with counts of specific cell types correlating with injury severity. Critically, novel usage of this method provided the first characterization of acute and chronic cellular inflammation after SCI to include a complete time course for polymorphonuclear leukocytes (PMNs, neutrophils), macrophages/microglia, and T-cells over a period ranging from 2 hours to 180 days post-injury (dpi), identifying a surprising novel second phase of cellular inflammation. Thorough characterization of cellular inflammation using this method may provide a better understanding of neuroinflammation in the injured CNS, and reveal an important multiphasic component of neuroinflammation that may be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions for SCI.

Quantification of cellular inflammation in the injured/pathological CNS by flow cytometry is complicated by lipid/myelin debris that can have similar size and granulation to cells, decreasing sensitivity/accuracy. We have advanced a cell preparation method to remove myelin debris and improve cell detection by flow cytometry in the injured spinal cord.

免疫细胞在受伤的脊髓组织流式细胞仪定量评估：一个细胞的分离纯化方法的新颖使用

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true ...

科研

免疫与感染

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis3 have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture.
Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions4. Radiation pressure was first observed and applied to optical tweezer systems in 19705, 6, and was first used to control biological specimens in 19877. Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena8-13.
We describe a method14 that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals15, 16 (e.g. AIDS, chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.

动态活细胞成像的宿主 - 病原体相互作用的研究使用的光学陷阱

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

Nontypeable Haemophilus influenzae (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory mucosa 1; 2. To study the mechanisms by which these organisms survive on and inside respiratory tissues, a model in which successful long-term co-culture of bacteria and human cells can be performed is required. We use primary human respiratory epithelial tissues raised to the air-liquid interface, the EpiAirway model (MatTek, Ashland, MA). These are non-immortalized, well-differentiated, 3-dimensional tissues that contain tight junctions, ciliated and nonciliated cells, goblet cells that produce mucin, and retain the ability to produce cytokines in response to infection. 
This biologically relevant in vitro model of the human upper airway can be used in a number of ways; the overall goal of this method is to perform long-term co-culture of EpiAirway tissues with NTHi and quantitate cell-associated and internalized bacteria over time. As well, mucin production and the cytokine profile of the infected co-cultures can be determined. This approach improves upon existing methods in that many current protocols use submerged monolayer or Transwell cultures of human cells, which are not capable of supporting bacterial infections over extended periods3. For example, if an organism can replicate in the overlying media, this can result in unacceptable levels of cytotoxicity and loss of host cells, arresting the experiment. The EpiAirway model allows characterization of long-term host-pathogen interactions. Further, since the source for the EpiAirway is normal human tracheo-bronchial cells rather than an immortalized line, each is an excellent representation of actual human upper respiratory tract tissue, both in structure and in function4.
For this method, the EpiAirway tissues are weaned off of anti-microbial and anti-fungal compounds for 2 days prior to delivery, and all procedures are performed under antibiotic-free conditions. This necessitates special considerations, since both bacteria and primary human tissues are used in the same biosafety cabinet, and are co-cultured for extended periods.

This method allows characterization of extended bacterial co-culture with EpiAirways, primary human respiratory epithelial tissue grown at the air-liquid interface, a biologically relevant in vitro model. The approach can be used with any microbe that is amenable to long-term co-culture.

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

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4&#39;,6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells.

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells.

荧光显微镜方法测定细菌在协会的可行性与哺乳动物细胞

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

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and frequently of indigenous origin. The ability of some anaerobic pathogens to invade human cells gives them adaptive measures to escape innate immunity as well as to modulate host cell behavior. However, ensuring that the anaerobic bacteria are live during experimental investigation of the events may pose challenges. Porphyromonas gingivalis, a Gram-negative anaerobe, is capable of invading a variety of eukaryotic non-phagocytic cells. This article outlines how to successfully culture and assess the ability of P. gingivalis to invade human umbilical vein endothelial cells (HUVECs). Two protocols were developed: one to measure bacteria that can successfully invade and survive within the host, and the other to visualize bacteria interacting with host cells. These techniques necessitate the use of an anaerobic chamber to supply P. gingivalis with an anaerobic environment for optimal growth.
The first protocol is based on the antibiotic protection assay, which is largely used to study the invasion of host cells by bacteria. However, the antibiotic protection assay is limited; only intracellular bacteria that are culturable following antibiotic treatment and host cell lysis are measured. To assess all bacteria interacting with host cells, both live and dead, we developed a protocol that uses fluorescent microscopy to examine host-pathogen interaction. Bacteria are fluorescently labeled with 2&#39;,7&#39;-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and used to infect eukaryotic cells under anaerobic conditions. Following fixing with paraformaldehyde and permeabilization with 0.2% Triton X-100, host cells are labeled with TRITC phalloidin and DAPI to label the cell cytoskeleton and nucleus, respectively. Multiple images taken at different focal points (Z-stack) are obtained for temporal-spatial visualization of bacteria. Methods used in this study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

This article presents two protocols: one to measure anaerobic bacteria that can successfully invade and survive within the host, and the other to visualize anaerobic bacteria interacting with host cells. This study can be applied to any cultivable anaerobe and any eukaryotic cell type.

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 ...

The Use of Flow Cytometry to Assess the State of Chromatin in T Cells

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to clonal proliferation of only those T cells that bear a receptor that recognizes the antigen. Chromatin decondensation is a hallmark of T cell activation and is required for T cells to acquire the ability to proliferate after antigen engagement. This change in chromatin condensation can be detected using antibodies raised against histone proteins. These antibodies cannot bind to their epitopes in na&#239;ve T cells as well as they can in activated T cells. We describe how to simultaneously stain T cell-specific surface markers, track viability with a fixable dead cell stain, and measure chromatin status via intracellular staining of Histone H3 proteins. Stained cells are analyzed by flow cytometry and chromatin condensation status is measured as the mean fluorescence intensity (MFI) of the Histone H3 stain. Chromatin decondensation during T cell activation is demonstrated as an increase in the MFI

Flow cytometry can be utilized to assess the state of chromatin within T cells. This protocol allows scientists to interpret evidence of chromatin decondensation during T cell activation demonstrated by an increase in the mean florescence intensity (MFI) of fluorescent Histone H3 antibodies

流式细胞仪用来评估染色质的T细胞国家

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

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular components responsible for supplying cell energy; however, excess mitochondria also produce reactive oxygen species (ROS) that could cause cell death. Therefore, the number of mitochondria must constantly be adjusted to fit the needs of the cells. This dynamic regulation is achieved in part through the function of lysosomes that remove surplus/damaged organelles and macromolecules. Hence, cellular mitochondrial and lysosomal contents are key indicators to evaluate the metabolic adjustment of cells. With the development of probes for organelles, well-characterized lysosome or mitochondria-specific dyes have become available in various formats to label cellular lysosomes and mitochondria. Multicolor flow cytometry is a common tool to profile cell phenotypes, and has the capability to be integrated with other assays. Here, we present a detailed protocol of how to combine organelle-specific dyes with surface markers staining to measure the amount of lysosomes and mitochondria in different T cell populations on a flow cytometer.

This article illustrates a powerful method to quantify mitochondria or lysosomes in living cells. The combination of lysosome- or mitochondria-specific dyes with fluorescently conjugated antibodies against surface markers allows the quantification of these organelles in mixed cell populations, like primary cells harvested from tissue samples, by using multicolor flow cytometry.

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

Obesity is associated with chronic low-grade inflammation and insulin resistance, contributing to an increasing prevalence of chronic metabolic diseases, such as type 2 diabetes and nonalcoholic steatohepatitis (NASH). Recent research has established that pro-inflammatory immune cells infiltrate obese hypertrophic adipose tissue and liver. Given the emerging importance of immune cells in the context of metabolic homeostasis, there is a critical need to quantify and characterize their modification during the development of type 2 diabetes and NASH. However, animal models that induce pathophysiological features typical of human NASH are sparse.
In this article, we provide a detailed protocol to identify immune cell subsets isolated from liver and adipose tissue in a reliable mouse model of NASH, established by housing high-fat diet (HFD) mice under non-specific pathogen-free (SPF) conditions without a barrier for at least seven weeks. We demonstrate the handling of mice in non-SPF conditions, digestion of the tissues and identification of macrophages, natural killer (NK) cells, dendritic cells, B and T cell subsets by flow cytometry. Representative flow cytometry plots from SPF HFD mice and non-SPF mice are provided. To obtain reliable and interpretable data, the use of antibodies, accurate and precise methods for tissue digestion and proper gating in flow cytometry experiments are critical elements.
The intervention to restore physiological antigen exposure in mice by housing them in non-SPF conditions and unspecific exposure to microbial antigens could provide a relevant tool for investigating the link between immunological alterations, diet-induced obesity and related long term complications.

A simple and reliable diet-induced rodent animal model for nonalcoholic steatohepatitis (NASH) is described, achieved through non-SPF housing of the animals and administration of a specific high-fat diet. We describe identification of hepatic and adipose immune cell subsets to recapitulate human immunological conditions by exposing mice to environmental germs.

非酒精性脂肪性肝炎合并2型糖尿病的一种先进的小鼠模型

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

Analysis of SAMHD1 Restriction by Flow Cytometry in Human Myeloid U937 Cells

Sterile &#945;-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) inhibits replication of HIV-1 in quiescent myeloid cells. U937 cells are widely used as a convenient cell system for analyzing SAMHD1 activity due to a low level of SAMHD1 RNA expression, leading to undetectable endogenous protein expression. Based on similar assays developed in the Stoye laboratory to characterize other retroviral restriction factors, the Bishop lab developed a two-color restriction assay to analyze SAMHD1 in U937 cells. Murine Leukaemia Virus-like particles expressing SAMHD1, alongside YFP expressed from an IRES, are used to transduce U937 cells. Cells are then treated with phorbol myristate acetate to induce differentiation to a quiescent phenotype. Following differentiation, cells are infected with HIV-1 virus-like particles expressing a fluorescent reporter. After 48 h, cells are harvested and analyzed by flow cytometry. The proportion of HIV-infected cells in the SAMHD1-expressing population is compared to that in internal control cells lacking SAMHD1. This comparison reveals a restriction ratio. SAMHD1 expression leads to a five-fold reduction in HIV infection, corresponding to a restriction ratio of 0.2. Our recent substitution of RFP for the original GFP as the reporter gene for HIV infection has facilitated flow cytometry analysis.
This assay has been successfully used to characterize the effect of amino acid substitutions on SAMHD1 restriction by transducing with viruses encoding altered SAMHD1 proteins, derived from site-directed mutagenesis of the expression vector. For example, the catalytic site substitutions HD206-7AA show a restriction phenotype of 1, indicating a loss of restriction activity. Equally, the susceptibility of different tester viruses can be determined. The assay can be further adapted to incorporate the effect of differentiation status, metabolic status, and SAMHD1 modifiers to better understand the relationship between SAMHD1, cell metabolic state, and viral restriction.

Described here is an established method to determine the extent of HIV-1 restriction by the cellular inhibitory protein SAMHD1. Human myeloid lineage U937 cells are transduced with a SAMHD1 expression vector co-expressing YFP, differentiated and then challenged with HIV-RFP. The level of restriction is determined by flow cytometry analysis.

流式细胞术分析人髓系U937细胞中SAMHD1限制性

Sterile &#945;-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) inhibits replication of HIV-1 in quiescent myeloid cells. U937 cells are ...

An Automated Culture System for Use in Preclinical Testing of Host-Directed Therapies for Tuberculosis

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), was the most significant infectious disease killer globally until the advent of COVID-19. Mtb has evolved to persist in its intracellular environment, evade host defenses, and has developed resistance to many anti-tubercular drugs. One approach to solving resistance is identifying existing&#160;approved drugs that will boost the host immune response to Mtb. These drugs could then be repurposed as adjunctive host-directed therapies (HDT) to shorten treatment time and help overcome antibiotic resistance.
Quantification of intracellular Mtb growth in macrophages is a crucial aspect of assessing potential HDT. The gold standard for measuring Mtb growth is counting colony-forming units (CFU) on agar plates. This is a slow, labor-intensive assay that does not lend itself to rapid screening of drugs. In this protocol, an automated, broth-based culture system, which is more commonly used to detect Mtb in clinical specimens, has been adapted for preclinical screening of host-directed therapies. The capacity of the liquid culture assay system to investigate intracellular Mtb growth in macrophages treated with HDT was evaluated. The HDTs tested for their ability to inhibit Mtb growth were all-trans Retinoic acid (AtRA), both in solution and encapsulated in poly(lactic-co-glycolic acid) (PLGA) microparticles and the combination of interferon-gamma and linezolid. The advantages of this automated liquid culture-based technique over the CFU method include simplicity of setup, less labor-intensive preparation, and faster time to results (5-12 days compared to 21 days or more for agar plates).

Rapid and efficient quantification of intracellular M. tuberculosis growth is crucial for pursuing improved therapies against tuberculosis (TB). This protocol describes a broth-based colorimetric detection assay using an automated liquid culture system to quantify Mtb growth in macrophages treated with candidate host-directed therapies.

用于宿主靶向治疗结核病临床前检测的自动化培养系统

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), was the most significant infectious disease killer globally until the advent ...