Name: induction of eryptosis in red blood cells using a calcium ionophore
Uploaded: 2020-01-21T15:00:00
Description: Watch this Scientific Journal Video about Induction of Eryptosis in Red Blood Cells Using a Calcium Ionophore at JoVE.com

这项工作得到了NIH授予R15ES030140和NSF授予CBET1903568的支持。来自罗斯工程技术学院和俄亥俄州立大学化学和生物分子工程系的财政支持也得到了认可。

下文所述协议中使用的所有人体血液样本均作为去身份化样本购买。没有人类受试者直接参与或招募参与这项研究。在涉及人类研究时，应使用《赫尔辛基宣言》的指导方针。
1. 从全血分离的红细胞
<ol><li>将500μL全血加入酸锡酸盐Dextrose（ACD）（储存在4°C）到微离心管中。 注：全血是在ACD中购买的。据该公司称，1.5 mL的ACD被添加到7 mL的全血（8.5 mL总体积）。</li>
	<li>在?...

<ol>
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此过程的目标是为电离磷浓度、治疗时间和细胞外葡萄糖浓度提供最佳值，这是确保成功诱导红斑气的重要因素。该协议的一个关键步骤是细胞外葡萄糖的耗竭，尽管其重要性，但文献中并未充分强调这一点。正常环状溶液（5 mM）中的糖含量对红斑酶有抑制作用。细胞外环境中的葡萄糖消耗会诱发细胞压力并激活蛋白激酶C（PKC），从而激活钙和钾通道。这导致Ca2+在细胞体空间30，31，34?...

红细胞中的程序性细胞死亡，也称为红斑病，在许多临床条件和血液学疾病中很常见。脂质增，与细胞收缩和细胞血浆膜1、2磷脂不对称的丧失有关。不对称性丧失导致磷脂酰丝氨酸（PS）的易位，这种脂质通常位于内侧传单3，4，到细胞外传单，信号到巨噬细胞到噬菌体和去除有缺陷的红细胞5，6，7，8。在红细胞正常寿命结束时，通过巨噬细胞去除红细胞可确保循环中的红细胞平衡。然而，在疾病条件下，如刀状细胞疾病和地中海贫血9，10，11，增强性红斑狼疮可能导致严重的贫血2。由于其在血液病学中的重要性，研究诱导或抑制红斑病的因素以及这一过程背后的分子机制具有极大的兴趣。
健康红细胞的血浆膜是不对称的，不同的磷脂在外部和内侧分布。膜不对称主要受膜酶作用的调节。氨磷脂转氯酶通过将这些脂质引导到细胞内部传单，促进氨磷脂、PS和磷脂酰胺（PE）的运输。另一方面，软体酶将含有磷脂、磷脂酰胆碱（PC）和斯芬戈米林（SM）的胆碱从细胞膜的内侧输送到外膜12。然而，与健康细胞不同，红斑红细胞的膜被搅乱。这是由于第三种酶，scramblase的作用，它通过促进氨磷脂的双向传输13，14，15，16破坏磷脂不对称。斯兰布拉斯由Ca2+细胞内水平升高激活。因此，促进Ca2+在细胞膜12间运输的电泳钙是红斑狼疮的有效诱因。
碘霉素，一种电磷钙，已广泛用于诱导红细胞12，17，18，19，20，21，22，23，24，25，26的红斑狼疮。碘霉素同时具有亲水性和疏水性组，这些组是结合和捕获Ca2+离子，并将其输送到细胞空间27、28、29所必需的。这导致scramblase的激活和PS的易位到外传单，这可以很容易地检测使用附件-V，一种细胞蛋白与PS12高度亲和力。虽然通常报道用碘霉素引发红斑狼疮，但文献中存在相当大的方法差异（表1）。经历红斑狼疮的红细胞群取决于不同的因素，如电离度浓度、电离磷的治疗时间以及细胞外环境的糖含量（葡萄糖消耗激活阳离子通道，并便利Ca2+进入细胞空间）30，31。然而，这些因素在文献中几乎没有一致性，使得在体外难以进行红斑狼疮的排斥性。
在这个协议中，我们提出了一个分步程序，以诱导红细胞中的红细胞。检查影响成功红斑狼疮的因素，包括Ca2+浓度、电离浓度、治疗时间和葡萄糖耗尽缓冲液的预孵育，并报告最佳值。此过程表明，与含葡萄糖缓冲液相比，无葡萄糖缓冲液中红细胞的预孵育可显著增加红细胞分百分比。该协议可用于实验室生产各种应用的红细胞红细胞。

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			<td>96-well plate</td>
			<td>Fisher Scientific</td>
			<td>12-565-331</td>
			<td></td>
		</tr>
		<tr>
			<td>Annexin V Alexa Fluor 488 - apoptosis kit</td>
			<td>Fisher Scientific</td>
			<td>A10788</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>BD FACSAria II flow cytometer</td>
			<td>BD Biosciences</td>
			<td>643177</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Fisher Scientific</td>
			<td>C79-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Millipore Sigma</td>
			<td>M7157</td>
			<td>Model Eppendorf 5415C</td>
		</tr>
		<tr>
			<td>Confocal fluorescence microscopy</td>
			<td>Zeiss, LSM Tek Thornwood</td>
			<td></td>
			<td>Model LSM 510, Argon laser excited at 488 nm for taking images</td>
		</tr>
		<tr>
			<td>Cover glasses circles</td>
			<td>Fisher Scientific</td>
			<td>12-545-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable round bottom flow cytometry tube</td>
			<td>VWR</td>
			<td>VWRU47729-566</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>472301-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>VWR Life Science</td>
			<td>SH30028.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose monohydrate</td>
			<td>Sigma-Aldrich</td>
			<td>Y0001745</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES Buffer (1 M)</td>
			<td>Fisher Scientific</td>
			<td>50-751-7290</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Ionomycin calcium salt</td>
			<td>EMD Milipore Corp.</td>
			<td>407952-1MG</td>
			<td>Dissolve in DMSO to reach 2 mM. Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Fisher Scientific</td>
			<td>P330-500</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Fisher Scientific</td>
			<td>M65-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tube</td>
			<td>Fisher Scientific</td>
			<td>02-681-5</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher Scientific</td>
			<td>S271-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Plain glass microscope slides</td>
			<td>Fisher Scientific</td>
			<td>12-544-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Synergy HFM microplate reader</td>
			<td>BioTek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Whole blood in ACD</td>
			<td>Zen-Bio</td>
			<td></td>
			<td>Store at 4 &#176;C and warm to 37 &#176;C prior to use</td>
		</tr>
	</tbody>
</table>

induction of eryptosis in red blood cells using a calcium ionophore

红细胞病，红细胞程序性细胞死亡，发生在一些血液学疾病和红细胞损伤期间。红斑细胞的一个特征是细胞膜成分不对称的丧失，导致磷脂酰丝氨酸在膜外膜上的易位。这个过程是由Ca2+细胞内浓度的增加触发的，它激活scramblase，一种促进膜传单之间磷脂双向运动的酶。鉴于红斑病在各种疾病条件下的重要性，已经努力在体外诱导红斑狼疮。这些努力通常依靠电离钙，碘霉素，以提高细胞内Ca2+浓度和诱导红斑。然而，在文献中报告了许多关于使用碘霉素诱导红斑狼疮的程序的差异。在此，我们报告人类红细胞中碘霉素引起的红斑狼疮的分步方案。我们专注于该程序的重要步骤，包括电离物浓度、孵育时间和葡萄糖消耗，并提供具有代表性的结果。该协议可用于在实验室中可重复诱导红斑狼疮。

碘霉素浓度的优化
虽然用碘霉素来诱发红斑狼疮，但增加的碘霉素浓度可导致溶血（即红细胞的溶化和血红蛋白的释放），需要避免。在林格溶液中用1μM电霉素治疗红细胞2小时足以诱发红斑狼疮，通过FACS分析（图1A）成功贴标附件-V Alexa面粉488结合和定量就证明了这一点。碘霉...

Watch this Scientific Journal Video about Induction of Eryptosis in Red Blood Cells Using a Calcium Ionophore at JoVE.com

Induction of Eryptosis in Red Blood Cells Using a Calcium Ionophore

提供了一种诱导红斑病的方案，即红细胞中程序化细胞死亡，使用碘化钙，碘霉素。通过监测膜外传单中的局部磷脂酰丝氨酸来评估成功的红斑狼疮。已经审查了影响协议成功的因素，并提供了最佳条件。

使用碘电磷诱导红血球中的红血球

Eryptosis, erythrocyte programmed cell death, occurs in a number of hematological diseases and during injury to erythrocytes. A hallmark of eryptotic ...

有许多努力使用异霉素诱导红斑病。然而，文献中并没有清楚地概述这一过程的确切程序。我们提供一个分步程序，使用异霉素诱导红斑病。该程序的主要优点是以可靠和可重复的方式诱导人类红细胞的红斑病。首先，在酸性柠檬酸葡萄糖中加入500微升的冷全血，加入微离心管。在室温下以700倍g离心，将整个血液离心5分钟，并使用移液器去除透明血浆和薄薄的薄薄外衣，以离开红红细胞层。准备一升含有125毫摩尔氯化钠、5毫摩尔氯化钾、1毫摩尔硫酸镁、32毫摩尔性海苯、5毫摩尔葡萄糖和1毫摩尔氯化钙的环果溶液。通过添加两滴微升水杨氢氧化钠，将 pH 调整为 7.4。要准备无葡萄糖环线溶液，请遵循相同的协议，但不包括葡萄糖在溶液中。在林格溶液中清洗红细胞两次，将细胞颗粒悬浮在1.5毫升的林格溶液中，在室温下以700倍g离心5分钟，并去除上光。然后，在9，960微升无葡萄糖环环溶液中重新吸收40微升红细胞颗粒，使0.4%的血红素达到10毫升的最终体积。在37摄氏度下孵育细胞悬浮液7天。在无葡萄糖缓冲液中预孵育红细胞可显著触发红斑病。在无糖缓冲液中经过七天的预孵化后，获得了高红斑病。在DMSO的630微升中溶解一毫克离子霉素钙盐，达到2毫摩尔的最终浓度。在20毫升内，储存在零下20摄氏度。服用一毫升的0.4%造血剂，并加入0.5微升的两毫摩尔类霉素，以达到一微摩尔的最终浓度。在37摄氏度下孵育两小时。使用一毫升的血红素，没有离子素治疗作为负对照。在室温下将经过700次处理和未经处理的造血素离心5分钟，并去除其超钠，将细胞颗粒留在管底。使用振铃溶液将细胞颗粒悬浮在 1.5 毫升的振铃溶液中，在室温下以 700 倍 g 离心 5 分钟，并丢弃超钠，用振铃溶液清洗细胞三次。为了测量造血，将未经处理的0.4%造血剂中加入1毫升，加入微离心管，在37摄氏度下孵育两小时，作为0%血解的负对照。为了准备100%血解的阳性控制，在室温下以700倍g的微离心管和离心机中加入未经处理的0.4%血红素的一毫升，5分钟。取出上一提液，在细胞颗粒中加入一毫升蒸馏水，在37摄氏度下重新暂停和孵育两小时。接下来，在微离心管中加入一毫升处理0.4%血红素的离子霉素。将未经处理的细胞、经过处理的细胞和蒸馏水中的细胞以700倍g离心，在室温下5分钟。将 200 微升的超糖转移到 96 井板的每个井中。使用微板读卡器测量 541 纳米的吸光度。使用 A0、A100 和 AT 是水中林格溶液中红细胞的吸收度和离子霉素处理过的二核细胞的吸光度等公式计算血解。将 5X 附件-V 结合缓冲液中的两毫升稀释为 8 毫升 PBS，以获得 1X 结合缓冲液。将未经处理的离子素处理和未经处理的细胞颗粒在1X结合缓冲液的一毫升中重新释放。在结合缓冲液中加入235个细胞悬浮液，并在微离心管中加入15微升附件-V Alexa Fluor 488结合。在室温下在黑暗的地方孵育细胞20分钟。在室温下以700倍g离心5分钟。拆下上一液。将细胞颗粒悬浮在1.5毫升的结合缓冲液中，并在室温下以700倍g离心5分钟，用1X结合缓冲液清洗细胞两次。去除上流剂，将细胞颗粒重新泵中 250 微升 1X 结合缓冲液中，用于流量细胞测量。现在，将附件-V染色红细胞的200微升转移到一毫升圆底聚苯乙烯管中，该管与流式细胞仪兼容。登录到流式细胞学软件，然后单击新的实验按钮。单击新的管按钮。选择全局表并选择应用分析，以测量激发波长为 488 纳米、发射波长为 530 纳米的荧光强度。将要收集的细胞数设置为 20，000 个，用于荧光激活细胞分选分析。选择所需的管子并单击加载按钮。单击记录按钮进行向前散射和侧散射测量。对所有样本重复所有示例。右键单击试样按钮，然后单击应用批次分析生成结果文件。右键单击试样按钮，然后单击生成 FSC 文件。在该协议中，用一微摩尔离子素和林格溶液治疗红细胞两个小时足以诱发红斑病，通过FACS分析成功用附件-V氟488结合和定量标记就证明了红斑酸。5 和 10 微摩尔的异霉素浓度较高，导致红斑病略有增加。然而，这种浓度也加强了血解。在离子霉素和林格溶液中孵育红细胞的时间只有30分钟，足以诱发红斑病。孵育时间增加，使红斑病水平增加长达两个小时。然而，进一步孵育时间导致红斑病水平略有下降。180分钟后血解的较高值解释了在用异霉素治疗180分钟后，与存活较少的细胞相同数量的孵化后，红斑病的减少。具有离子霉素处理的红细胞在外叶中与磷脂酰丝氨酸结合时显示出明亮的荧光信号。相比之下，没有治疗的细胞显示非常弱的荧光信号，表明非常低的红斑病。在无葡萄糖缓冲液中预孵育是诱导性红斑病的重要诱因。由于在多种疾病中观察到性病，因此遵循此协议的实验是场特定的。在我们的实验室中，我们有兴趣研究红斑病对膜生物物理特性和膜与纳米材料相互作用的影响。Eryptosis 与大量疾病有关，包括糖尿病、刀状细胞贫血和地中海贫血。拥有诱导红斑病的可重复方案可以帮助这些领域的研究人员进一步研究每个领域特有的科学问题。

Research

JoVE Journal

Biochemistry

Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells

线粒体钙在分离线粒体和培养细胞中的流入分析

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ...

科研

生物化学

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

用石英谐振器研究磷脂结合蛋白附件 a2 与固体支持的脂质层结合动力学的耗散微重法

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

在 Vivo 钙成像中，在 C. elegans身体壁肌肉

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for ...

Translating Ribosome Affinity Purification (TRAP) for RNA Isolation from Endothelial Cells In Vivo

Translating Ribosome Affinity Purification TRAP for RNA Isolation from Endothelial Cells In Vivo

翻译核糖体亲性纯化(TRAP),用于从体内内皮细胞中分离RNA

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis ...

SUMO-Binding Entities (SUBEs) as Tools for the Enrichment, Isolation, Identification, and Characterization of the SUMO Proteome in Liver Cancer

SUMO-Binding Entities SUBEs as Tools for the Enrichment, Isolation, Identification, and Characterization of the SUMO Proteome in Liver Cancer

SUMO 结合实体 （SUBEs） 作为肝癌中 SUMO 蛋白酶的浓缩、分离、识别和表征的工具

Post-translational modification is a key mechanism regulating protein homeostasis and function in eukaryotic cells. Among all ubiquitin-like proteins in ...

Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology

使用免疫增强技术的超高速西布洛特

A western blot (also known as an immunoblot) is a canonical method for biomedical research. It is commonly used to determine the relative size and ...

Rapid Determination of Antibody-Antigen Affinity by Mass Photometry

通过质量光度法快速测定抗体抗原亲和力

Measurements of the specificity and affinity of antigen-antibody interactions are critically important for medical and research applications. In this ...

Inner Mitochondrial Membrane Sensitivity to Na+ Reveals Partially Segmented Functional CoQ Pools

Inner Mitochondrial Membrane Sensitivity to Na+ Reveals Partially Segmented Functional CoQ Pools

线粒体内膜对Na+ 的敏感性揭示了部分分割的功能性辅酶Q库

Ubiquinone (CoQ) pools in the inner mitochondrial membrane (IMM) are partially segmented to either complex I or FAD-dependent enzymes. Such subdivision ...

A Semi-Automated and Reproducible Biological-Based Method to Quantify Calcium Deposition In Vitro

A Semi-Automated and Reproducible Biological-Based Method to Quantify Calcium Deposition In Vitro

一种半自动且可重现的基于生物学的体外定量钙沉积的方法

Vascular calcification involves a series of degenerative pathologies, including inflammation, changes to cellular phenotype, cell death, and the absence ...

A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors

用于重组G蛋白偶联受体高通量筛选的“双加法”钙荧光测定

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening ...