| Isolation of Murine Alveolar Type II Epithelial Cells
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Author:
Date:
2017-05-20
[Abstract] We have optimized a protocol for isolation of alveolar type II epithelial cells from mouse lung. Lung cell suspensions are prepared by intratracheal instillation of dispase and agarose followed by mechanical disaggregation of the lungs. Alveolar type II epithelial cells are purified from these lung cell suspensions through magnetic-based negative selection using a Biotin-antibody, Streptavidin-MicroBeads system. The purified alveolar type II epithelial cells can be cultured and maintained on fibronectin-coated plates in DMEM with 10% FBS. This protocol enables specific investigation of alveolar type II epithelial cells at molecular and cellular levels and provides an important tool to investigate in vitro the mechanisms underlying lung pathogenesis.
[摘要] 我们优化了从小鼠肺分离肺泡II型上皮细胞的方案。通过气管内滴注分泌酶和琼脂糖，然后机械解聚肺来制备肺细胞悬浮液。通过使用生物素抗体Streptavidin-MicroBeads系统的磁性阴性选择，从这些肺细胞悬液中纯化肺泡II型上皮细胞。可以将纯化的肺泡II型上皮细胞培养并维持在含有10％FBS的DMEM中的纤连蛋白包被的平板上。该方案能够在分子和细胞水平上对肺泡II型上皮细胞进行特异性研究，并提供了一种重要的工具，用于在体外研究肺发病机制的机制。
背景 肺泡II型上皮细胞在肺泡完整性维持，表面活性蛋白合成和分泌中起关键作用，并防止细菌和病毒的肺部感染。最近使用小鼠肺癌模型的研究已经证明，肺泡II型上皮细胞是由化学致癌物质和致癌突变诱导的腺瘤/腺癌的关键细胞（Qu 等人，2015; Zhou > et al。，2015和2017）。为了进一步扩大我们对肺泡II型上皮细胞在体内肺发病机制中的作用的理解，需要分离肺泡II型上皮细胞以允许体外精确的机理分析， EM>。基于先前的研究（Corti等人，1996; Rice等人，2002），在我们的实验室中使用了一种修饰的方法来分离高度纯化的，可行的和可培养的来自小鼠的肺泡II型上皮细胞（Zhou等人，2015; Sun等人，2016）。
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| Immunoplaque Assay (Influenza Virus)
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Author:
Date:
2013-11-05
[Abstract] Despite developed long time ago, plaque assay is still the gold standard for viral titer quantification in modern virology. The standard crystal violet-based plaque assay relies on virus’ ability to induce cytopathic effect (CPE) which limits the assay to lytic viruses. Alternative viral quantification assays such as 50% tissue culture infectious assay (TCID50) and genetic material quantification by Q-PCR provide a different way of viral quantification with their own shortcoming. In here, we modified the fluorescent focus assay and developed an antibody-based immunoplaque assay which provides a reliable and reproducible viral quantification independent of CPE. Our assay not only allows accurate determination of viral titer, but also provides information on viral kinetics, ...
[摘要] 尽管发展很久以前，斑块测定仍然是现代病毒学病毒滴度量化的黄金标准。 标准的基于结晶紫的斑块测定依赖于病毒诱导细胞病变效应（CPE）的能力，其限制了对裂解病毒的测定。 替代性病毒定量测定如50％组织培养感染测定（TCID 50）和通过Q-PCR的遗传物质定量提供了具有其自身缺点的病毒定量的不同方式。 在这里，我们修改荧光焦点测定和开发基于抗体的免疫斑检测提供可靠和可重复的病毒定量独立于CPE。 我们的测定不仅允许病毒滴度的精确测定，而且提供关于病毒动力学，遗传稳定性和病毒种群的纯度的信息。
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| Neutral Red Assay for Murine Norovirus Replication and Detection in a Mouse
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Author:
Date:
2013-04-05
[Abstract] Neutral red (NR) is a dye that must be actively imported into the cell, and, therefore, the dye has been used for decades to selectively stain living cells. In addition, NR can also be incorporated into virus particles, although the mechanism behind this is poorly understood. Once encapsulated into the virion, NR, a light sensitive dye, can be photoactivated to inactivate the virus. The proposed mechanism explaining this observation is that activation of NR allows the dye to cross-link viral genome to viral capsid and thus preventing viral uncoating and infection. To study the early events of murine norovirus (MNV)-host interaction, light-sensitive NR-containing MNV is used to distinguish between input virus (i.e., NR-containing virus) and replicated virus (i.e., ...
[摘要] 中性红（NR）是必须主动导入细胞的染料，因此，染料已经使用数十年来选择性地染色活细胞。此外，NR也可以被并入病毒颗粒中，虽然其背后的机制知之甚少。一旦包封入病毒粒子中，NR（一种光敏染料）可以被光活化以使病毒失活。解释这种观察的提出的机制是NR的活化允许染料将病毒基因组交联病毒衣壳，从而防止病毒脱壳和感染。为了研究鼠诺如病毒（MNV） - 宿主相互作用的早期事件，使用光敏感的含NR的MNV来区分输入病毒（即含有NR的病毒）和复制的病毒（ >即，无NR的病毒）。该方案描述了将NR掺入MNV衣壳中以及这些病毒颗粒用于通过标准噬菌斑测定在小鼠和组织培养物中检测病毒复制的用途。相同的技术也用于研究脊髓灰质炎病毒复制（1-3）。因此，存在该技术可用于另外的非包膜病毒的潜力。然而，这必须在逐案基础上进行测试，因为关于猫杯状病毒的未公开数据表明不是所有的病毒都能够将NR稳定地掺入其衣壳中（J.Parker，personal communication）。
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