| A 3D Skin Melanoma Spheroid-Based Model to Assess Tumor-Immune Cell Interactions
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Author:
Date:
2020-12-05
[Abstract] Three-dimensional (3D) tumor spheroids have the potential to bridge the gap between two-dimensional (2D) monolayer tumor cell cultures and solid tumors with which they share a significant degree of similarity. However, the progression of solid tumors is often influenced by the dynamic and reciprocal interactions between tumor and immune cells. Here we present a 3D tumor spheroid-based model that might shed new light on understanding the mechanisms of tumor and immune cell interactions. The model first utilizes the hanging drop assay, which serves as one of the simplest methods for generating 3D spheroids and requires no specialized equipment. Next, pre-established spheroids can be co-cultured either directly or indirectly with an immune cell population of interest. Using skin melanoma, we ...
[摘要] [摘要]三维（3D）肿瘤球体具有弥合二维（2D）单层肿瘤细胞培养物与实体瘤之间的差距的潜力，它们之间有着显着的相似性。然而，实体瘤的进展通常受肿瘤与免疫细胞之间的动态相互作用和相互影响的影响。在这里，我们提出了一个基于3D肿瘤球体的模型，该模型可能会为了解肿瘤与免疫细胞相互作用的机制提供新的思路。的该模型首先利用了悬滴法，这是生成3D球体的最简单方法之一，不需要专门的设备。接下来，可以将预先建立的球体与目标免疫细胞群体直接或间接共培养。使用皮肤黑色素瘤，我们提供了该模型的详细说明，这可能对成功治疗策略的开发具有重要意义。
[背景]三维（3D）肿瘤球体是球形的自组装肿瘤细胞聚集体，类似于微转移瘤并复制实体瘤的许多特征。就像在无血管实体瘤的非增生区域一样，球体内部区域的肿瘤细胞通常表现出扰动的基因和蛋白质表达，新陈代谢改变，细胞周期停滞和坏死（Sant and Johnston ，2017）。但是，用于生成3D椭球体的大多数当前可用技术是耗时，困难和昂贵的。一种简单，快速，简便的生成3D球体的方法是使用悬滴法（Foty ...
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| A Workflow for Ultra-rapid Analysis of Histone Post-translational Modifications with Direct-injection Mass Spectrometry
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Author:
Date:
2020-09-20
[Abstract] Chromatin modifications, like histone post translational modifications (PTMs), are critical for tuning gene expression and many other aspects of cell phenotype. Liquid chromatography coupled to mass spectrometry (LC-MS) has become the most suitable method to analyze histones and histone PTMs in a large-scale manner. Selected histone PTMs have known functions, and their aberrant regulation is linked to a wide variety of diseases, including cancer. However, histone analysis is scarcely used in diagnostics, partially due to the limited throughput and not ideal reproducibility of LC-MS based analysis. We describe a workflow that allows for high-throughput sample preparation is less than a day using 96-well plates. Following preparation, samples are sprayed into MS without LC, using an ...
[摘要] [抽象]像组蛋白翻译后修饰（PTM）一样，染色质修饰对于调节基因表达和细胞表型的许多其他方面至关重要。液相色谱-质谱联用（LC-MS）已成为最适合大规模分析组蛋白和组蛋白PTM的方法。选定的组蛋白PTM具有已知功能，其异常调节与包括癌症在内的多种疾病有关。但是，组蛋白分析很少用于诊断中，部分是由于通量有限且基于LC-MS的分析的重现性不理想。我们描述了一种使用96孔板进行少于一天的高通量样品制备的工作流程。制备后，使用自动直接进样（DI-MS）方法将样品喷雾到无LC的MS中。每次分析都可以通过45个PTM（甲基化，乙酰化和磷酸化（共151个组蛋白标记）和16个未修饰的组蛋白肽进行组蛋白变体的相对定量。由于没有残留或基于LC的批处理效应，该工作流程允许MS运行少于1分钟，并具有更高的重现性和耐用性。最后，我们描述了一种工程化的肽序列，用于精确监控样品制备的效率，可以在DI-MS运行期间检测到该效率。
[背景] 组蛋白是具有球形头部和N末端尾巴的碱性蛋白质，富含精氨酸和赖氨酸残基。一对典型的组蛋白H2A，H2B，H3和H4（称为核心组蛋白）形成一个八聚体，其周围147 ...
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| Multiplication and Growth Inhibition Activity Assays for the Zoonotic Malaria Parasite, Plasmodium knowlesi
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Author:
Date:
2020-09-05
[Abstract] Malaria remains a major cause of morbidity and mortality globally. Clinical symptoms of the disease arise from the growth and multiplication of Plasmodium parasites within the blood of the host. Thus in vitro assays to determine how drug, antibody and genetic perturbations affect the growth rate of Plasmodium parasites are essential for the development of new therapeutics and improving our understanding of parasite biology. As both P. falciparum and P. knowlesi can be maintained in culture with human red blood cells, the effect of antimalarial drugs and inhibitory antibodies that target the invasion or growth capacity of Plasmodium parasites are routinely investigated by using multiplication assays or growth inhibition activity (GIA) ...
[摘要] [摘要] 疟疾仍然是全球发病率和死亡率的主要原因。该疾病的临床症状源于宿主血液中疟原虫的生长和繁殖。因此，体外测定以确定药物，抗体和遗传扰动如何影响疟原虫寄生虫的生长速率对于开发新疗法和增进我们对寄生虫生物学的理解至关重要。由于两个恶性疟原虫和P. knowlesi 可以在培养物中维持与人体红细胞，抗疟疾药物和抑制性抗体靶向的侵袭能力的影响疟原虫寄生虫 是通过使用针对这两个物种乘法测定或生长抑制测定法常规地研究。该协议给出了详细的一步一步的过程来进行基于所述寄生虫乳酸脱氢酶的活性为基础的流式细胞仪乘法测定和生长抑制活性测定法测试性中和抗体的疟原虫knowlesi 适于人类红血细胞培养物中。虽然类似测定法是用于很好地建立的恶性疟原虫，P. knowlesi 被更密切相关的所有其他人类感染性物种（帕切科等人。，2018），因此可以用作替代用于测试药物和疫苗用于其它疟疾种类，例如如间日疟原虫，它是非洲以外疟疾最广泛的病因，但尚未在实验室条件下进行培养。
[背景 ] ...
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