| Simultaneous Intranasal/Intravascular Antibody Labeling of CD4+ T Cells in Mouse Lungs
|
|
Author:
Date:
2017-01-05
[Abstract] CD4+ T cell responses have been shown to be protective in many respiratory virus infections. In the respiratory tract, CD4+ T cells include cells in the airway and parenchyma and cells adhering to the pulmonary vasculature. Here we discuss in detail the methods that are useful for characterizing CD4+ T cells in different anatomic locations in mouse lungs.
[摘要] 许多呼吸道病毒感染中CD4细胞反应已被证明是保护性的。在呼吸道中，CD4 + T细胞包括气道中的细胞和附着于肺血管的细胞和细胞。在这里，我们详细讨论了可用于表征小鼠肺不同解剖位置的CD4 + T细胞的方法。
背景 为了区分循环和组织中的记忆T细胞，已经开发了一种T细胞血管内染色的方法（Anderson等人，2012）。这种方法被广泛用于定义许多器官和组织（包括肺，脾和肠）的记忆T细胞。然而，呼吸道中的记忆T细胞位于三个独特的解剖位置，即气道，薄壁组织和肺血管。血管内染色不能区分气道和实质中的细胞，因为它们都是从循环中分离的，血管内施用的抗体不会污染这两个群体。我们设计了同时进行的鼻内/血管内抗体标记测定，可以使用最少量的抗体来标记和区分所有三个位置的细胞。
|
|
|
| Determining Leukocyte Origins Using Parabiosis in the PyMT Breast Tumor Model
|
|
Author:
Date:
2015-08-20
[Abstract] Tumors develop in a complex microenvironment alongside numerous cell types that impact their survival. Immune cells make up a large proportion of these accessory cells and many are known to promote tumor progression. Macrophages, in particular, are associated with poor patient prognosis and are therefore potential candidates for therapeutic targeting in cancer. However, to develop successful strategies to target macrophages, it is important to clarify whether these cells are derived from blood-borne precursors or a tissue-resident population. Parabiosis, or the surgical connection of two mice resulting in a shared blood circulation, allows the distinction between these two cellular sources. Here, we describe the use of parabiosis to define cell ontogeny in a mouse model of breast cancer.
[摘要] 肿瘤在复杂的微环境中发展，伴随许多影响其存活的细胞类型。 免疫细胞构成这些附属细胞的大部分，并且已知许多细胞促进肿瘤进展。 巨噬细胞，特别是与不良的患者预后相关，因此是癌症中治疗靶向的潜在候选者。 然而，要开发成功的策略，以靶向巨噬细胞，重要的是要澄清这些细胞是否源自血源性前体或组织驻留人群。 Parabiosis或两个小鼠的外科连接导致共享的血液循环，允许这两个细胞来源之间的区别。 在这里，我们描述在乳腺癌的小鼠模型中定义细胞个体发育的拟杆菌的使用。
|
|
|
| Flow Cytometric Analysis of Autophagic Activity with Cyto-ID Staining in Primary Cells
|
|
Author:
Date:
2014-04-05
[Abstract] Flow cytometry allows very sensitive and reliable high-throughput analysis of autophagic flux. This methodology permits to screen cells in flow and capture multi-component images. Using this technology autophagic flux may be analysed accurately in both suspension as well as adherent cells upon trypsinization independent of how heterogeneous the autophagosomal content might be. The method is based on Cyto-ID staining of autophagic compartments (pre-autophagosomes, autophagosomes, and autophagolysosomes) in live cells using Cyto-ID® Autophagy Detection Kit. Autophagic compartments are intermediate constituents of a dynamic lysosomal degradation process and their intracellular abundance at a particular time point is a function of the established equilibrium between their ...
[摘要] 流式细胞术允许非常灵敏和可靠的高通量分析自噬通量。该方法允许在流动中筛选细胞并捕获多组分图像。使用这种技术，可以在悬浮液以及粘附细胞中在胰蛋白酶消化时精确分析自噬流，而与自噬体内容物的异质性无关。该方法基于使用Cyto-ID自动吞噬检测试剂盒的活细胞中的自体吞噬细胞（前自噬体，自噬体和自噬溶酶体）的Cyto-ID染色。自噬区室是动态溶酶体降解过程的中间组分，并且它们在特定时间点的细胞内丰度是其产生和降解之间建立的平衡的函数。自噬吞噬的确定有助于自噬体形成的早期诱导和自噬体成熟的晚期抑制之间的区分，因为两者都导致自噬体存在的最终增加。 Cyto-ID测定基于使用选择性染色自噬隔室的特定染料，因此允许测定自噬通量，作为染色隔室在碱性或活化条件[雷帕霉素（1-5μmol/L），PP242（1- （NH 4 Cl）（10-20μmol/L）或含有6mmol/L葡萄糖（饥饿培养基）的Hanks平衡盐溶液），使用溶酶体化合物阻断自噬溶酶体降解mmol/L）或氯喹（CQ）（5-10μmol/L）。 ΔMFICyto-ID = MFI Cyto-ID（+ CQ/NH 4 Cl）-MFI Cyto-ID（-CQ/NH 4 Cl）。
|
|
|