| Isolation of Mononuclear Cell Populations from Ovarian Carcinoma Ascites
|
|
Author:
Date:
2017-04-05
[Abstract] Ovarian cancer is one of the most fatal tumors in women. Due to a lack of symptoms and adequate screening methods, patients are diagnosed at advanced stages with extensive tumor burden (Jelovac and Armstrong, 2011). Interestingly, ovarian cancer metastasis is generally found within the peritoneal cavity rather than other tissues (Lengyel, 2010; Tan et al., 2006). The reason behind this tissue tropism of the peritoneal cavity remains elusive. A prominent feature of this selectivity is ascites, the accumulation of fluid within the peritoneal cavity, containing, amongst others, immune cells, tumor cells and various soluble factors that can be involved in the progression of ovarian cancer (Kipps et al., 2013). The protocol described here is used to isolate mononuclear cells ...
[摘要] 卵巢癌是女性最致命的肿瘤之一。由于缺乏症状和适当的筛查方法，患者被诊断为具有广泛肿瘤负担的晚期阶段（Jelovac和Armstrong，2011）。有趣的是，卵巢癌转移通常发生在腹腔内，而不是其他组织（Lengyel，2010; Tan等人，2006）。腹膜腔组织向性背后的原因仍然难以捉摸。这种选择性的一个突出特征是腹水，腹膜内的液体积累，其中包含免疫细胞，肿瘤细胞和可参与卵巢癌进展的各种可溶性因子（Kipps等，。，2013）。这里描述的方案用于从腹水中分离单核细胞以研究腹膜腔内免疫系统的功能。
使用Lymphoprep 的梯度离心是分离外周血单核细胞（PBMC）的标准方案。我们稍微调整了方案，关于样品制备和洗涤步骤的数量，以便从腹水中分离单核细胞。
|
|
|
| Ear Inflammation and Whole-mount Ear Staining
|
|
Author:
Date:
2016-10-20
[Abstract] The recruitment of circulating neutrophils from the bloodstream to the site of inflammation represents one of the earliest events during an innate immune response. During this response, neutrophils tether and roll along the vessel walls before transmigrating across the endothelium into the interstitial space to exert their functions. Here, we describe a protocol for the staining of intravascular and tissue-localized neutrophils following contact sensitization of the skin with croton oil. Visualization of the neutrophilic distribution in skin provides for a better interpretation of the local immune response.
[摘要] 循环嗜中性粒细胞从血流中募集到炎症部位代表先天免疫反应期间的最早期事件之一。在这种反应期间，嗜中性粒细胞在通过内皮迁移到间质空间以发挥其功能之前沿血管壁束缚和滚动。在这里，我们描述染色的血管内和组织定位中性粒细胞皮肤与巴豆油接触致敏后的协议。皮肤中嗜中性粒细胞分布的可视化提供了局部免疫应答的更好解释。
[背景] 炎症后中性粒细胞分布在皮肤中的表征代表了理解其专门功能的重要途径。即使通过流式细胞术广泛表征嗜中性粒细胞在炎症皮肤中的募集（Hampton等人，2015; Stock em 。，2014），这种技术提供有限的洞察中性粒细胞的血管内与间质定位。再循环和组织定位中性粒细胞展示不同的表型和功能，这使得他们的歧视需要识别当地免疫反应的关键参与者。随着活体显微镜（IVM）的发展，使得荧光标记的免疫细胞在体内的直接可视化成为可能。然而，由于IVM的高成本，这种强大的成像工具对研究人员的可访问性是有限的。在这里，我们提出了高分辨率静电成像的中性粒细胞在皮肤中使用成本效益试剂和通常可用的共聚焦激光扫描显微镜的替代协议。
|
|
|
| Determination of Cellular Phosphatidylinositol-3-phosphate (PI3P) Levels Using a Fluorescently Labelled Selective PI3P Binding Domain (PX)
|
|
Author:
Date:
2016-08-20
[Abstract] The lipid Phosphatidylinositol-3-phosphate [PtdIns3P or PI(3)P] plays many membrane trafficking roles and is primarily produced by the Class III PI3K, VPS34. Determining the level of cellular PI(3)P however can be complex. Extraction of cellular lipids by methanol/chloroform can struggle to separate and identify distinct phospholipid species. Alternately mass spectrometry may be utilised but this requires significant set up of specialised equipment and time to utilise.
Use of a PI(3)P-binding-specific recombinant protein domain is a quick method for ascertaining cellular PI(3)P levels and can also allow visualisation of sub-cellular localisation. The PX domain of p40phox (herein referred to as PX) is very specific for PI(3)P over other phospholipid species (Kanai et al., ...
[摘要] 脂质磷脂酰肌醇-3-磷酸[PtdIns3P或PI（3）P]扮演许多膜贩运角色，主要由III类PI3K，VPS34产生。然而，确定细胞PI（3）P的水平可以是复杂的。通过甲醇/氯仿提取细胞脂质可能难以分离和鉴定不同的磷脂种类。或者可以使用质谱法，但这需要大量设置专门的设备和时间来利用。
使用PI（3）P结合特异性重组蛋白结构域是用于确定细胞PI（3）P水平的快速方法，并且还可以允许亚细胞定位的可视化。 p40phox的PX结构域（本文称为PX）对PI（3）P比其它磷脂种类非常特异（Kanai等人，2001）。然而，在细胞中直接表达PX可能是有问题的，因为它将以显性负性方式作用，以比内源蛋白更大的亲和力结合和螯合PI（3）P，从而干扰细胞途径和PI（3）P水平。因此，使用荧光标记的PX后细胞固定更合适，因为它能突出显示PI（3）富集结构，没有扰乱系统的风险。
|
|
|