| Stopped-flow Light Scattering Analysis of Red Blood Cell Glycerol Permeability
|
|
Author:
Date:
2020-08-20
[Abstract] Stopped-Flow Light Scattering (SFLS) is a method devised to analyze the kinetics of fast chemical reactions that result in a significant change of the average molecular weight and/or in the shape of the reaction substrates. Several modifications of the original stopped-flow system have been made leading to a significant extension of its technical applications. One of these modifications allows the biophysical characterization of the water and solute permeability of biological and artificial membranes.
Here, we describe a protocol of SFLS to measure the glycerol permeability of isolated human red blood cells (RBCs) and evaluate the pharmacokinetics properties (selectivity and potency) of isoform-specific inhibitors of AQP3, AQP7 and AQP9, three mammalian aquaglyceroporins ...
[摘要] [摘要] 停流光散射（SFLS）是一种用于分析快速化学反应动力学的方法，该化学反应导致平均分子量和/或反应底物形状发生显着变化。已对原始停流系统进行了几处修改，从而大大扩展了其技术应用范围。这些修饰之一允许水的生物物理表征以及生物膜和人造膜的溶质渗透性。
在这里，我们描述了一种SFLS协议，用于测量分离的人红细胞（RBC）的甘油渗透性，并评估AQP3，AQP7和AQP9的同种型特异性抑制剂的药代动力学特性（选择性和效价），这三种哺乳动物的水甘油糖蛋白允许转运甘油跨膜。在20°C的SFLS设备中，将RBC的悬浮液（1％的血细胞比容）暴露于100 mM甘油的向内定向，并在530 nm的单色波长下记录120 s的散射光强度变化。该设备的死区时间为1.6毫秒，混合效率在不到1毫秒的时间内达到99％。数据拟合到单指数函数和有关的时间constan 吨（ ，对应于伴随甘油的进入红细胞的水渗透运动的光散射的细胞膨胀相的秒）进行测定。RBC 的甘油渗透系数（P gly ，cm / s）通过以下公式计算：
P gly = 1 / [[（（S / V） ]
其中 （s）是拟合的指数时间常数，S / V是分析的RBC 样本的表面积与体积之比（cm -1 ...
|
|
|
| Isolation and High Throughput Flow Cytometric Apoptosis Assay of Human Neutrophils to Enable Compound Library Screening
|
|
Author:
Date:
2020-06-05
[Abstract] The study of human neutrophils in vitro is challenging due to their short half-life and propensity for activation. However, with careful handling and manipulation in the laboratory, they can be a powerful tool to investigate immune responses in health and disease. Here we describe a method for the isolation of human neutrophils from peripheral blood samples, followed by a high-throughput screen to assess the efficacy of a library of compounds in inducing neutrophil apoptosis, which may have therapeutic potential in neutrophil-driven diseases. This protocol is based on previously-published neutrophil isolation methods utilizing Dextran sedimentation of red blood cells followed by the separation of granulocytes with plasma/Percoll discontinuous gradient centrifugation. Yields of ~1 ...
[摘要] [摘要] 人类嗜中性粒细胞的研究 由于其半衰期短且易于活化，因此体外具有挑战性，然而，通过在实验室中的仔细处理和操纵，它们可以成为研究健康和疾病中免疫反应的有力工具。在此，我们介绍了一种分离方法外周血样本中的人类嗜中性粒细胞，然后进行高通量筛选，以评估化合物库诱导嗜中性粒细胞凋亡的功效，该化合物在嗜中性粒细胞驱动的疾病中可能具有治疗潜力。此规程基于先前发表的嗜中性粒细胞分离方法利用红细胞的葡聚糖沉降，接着用等离子体/粒细胞的分离的Percoll 〜1×10个的不连续梯度centrifugation.Yields 6 每嗜中性粒细胞毫升的血液，和>的纯度95个％嗜中性粒细胞是典型的。嗜中性粒细胞与经处理的激酶抑制剂文库，然后通过流式细胞仪评估中性粒细胞凋亡的速率。低通量用于高通量筛选人类原代免疫细胞，以鉴定具有修饰嗜中性粒细胞功能的化合物，并可根据需要进行修饰以评估其他表型。
[背景] 中性粒细胞是重要的先天免疫细胞与键的角色防御抵抗infection.They是短暂的细胞中，在感染和inflammat部位延长6-8小时循环典型的半衰期离子（萨默斯等人。 。，2010 ; Hidalgo。Et Al ...
|
|
|
| Direct Reprogramming of Mouse Embryonic Fibroblasts to Conventional Type 1 Dendritic Cells by Enforced Expression of Transcription Factors
|
|
Author:
Date:
2020-05-20
[Abstract] Ectopic expression of transcription factor combinations has been recently demonstrated to reprogram differentiated somatic cells towards the dendritic cell (DC) lineage without reversion to a multipotent state. DCs have the ability to induce potent and long-lasting adaptive immune responses. In particular, conventional type 1 DCs (cDC1s) excel on antigen cross-presentation, a critical step for inducing CD8+ T cell cytotoxic responses. The rarity of naturally occurring cDC1s and lack of in vitro methodologies for the generation of pure cDC1 populations strongly hinders the study of cDC1 lineage specification and function. Here, we describe a protocol for the generation of induced DCs (iDCs) by lentiviral-mediated expression of the transcription factors PU.1, IRF8 and ...
[摘要] [摘要] 转录因子组合的异位表达最近被证明可以将分化的体细胞重编程为树突状细胞（DC）谱系，而不会回复到多能状态。DC具有诱导有效和持久的适应性免疫应答的能力。在特定的常规类型1的DC（cDC1s）练成抗原交叉呈递，用于诱导CD8的关键步骤+ T细胞的细胞毒性应答。天然存在的cDC1的稀有性和缺乏用于生成纯cDC1群体的体外方法论，严重阻碍了对cDC1谱系规格和功能的研究。在这里，我们描述了用于生成感应DC（iDC）的协议 慢病毒介导的转录因子PU.1，IRF8和BATF3在小鼠胚胎成纤维细胞中的表达。iDC 在9天内获得DC形态，cDC1表型和转录特征。使用此协议生成的iDC 具有对炎症刺激，吞噬死细胞，将抗原加工并交叉呈递给CD8 + T细胞的功能。DC重新编程提供了一个简单易处理的系统，可以生成大量的cDC1类细胞用于高内涵筛选，从而开辟了新途径，可以更好地了解cDC1的规格和功能。将来，在成纤维细胞中忠实诱导cDC1命运可能会导致产生患者特定的疫苗接种DC。
[背景技术树突状细胞（DC）是专业的抗原呈递细胞，专门用于识别，加工和呈递T细胞抗原，在诱导适应性免疫应答和免疫记忆中起关键作用（Me rad 等，2013）。DC可以分为4个主要子集：浆细胞样DC（pDC ），大量1型干扰素的产生者，循环单核细胞衍生的单核细胞衍生DC 和常规DC（cDC ...
|
|
|