Name: isolation and characterization of neutrophil-derived microparticles for functional studies
Uploaded: 2018-03-02T15:00:00
Description: Watch this Scientific Journal Video about Isolation and Characterization of Neutrophil-derived Microparticles for Functional Studies at JoVE.com

我们感谢 Suchitra Swaminathan 博士的技术援助, 他是西北范伯格医学院流式细胞术的核心。资助是由 (NIH) DK101675 提供的。

所有的动物工作都是由西北 IACUC 批准的。所有的实验都是按照所有相关的规章制度和制度准则完成的。对于献血者, 知情同意书被提出并签署;此外, 这项研究中的所有人类学科都是按照《机构和联邦人类福利准则》处理的。
注意: 协议步骤列在以下小节中: (i) 小鼠骨髓细胞分离;(ii) 中性粒细胞与小鼠骨髓隔绝;(iii) 中性粒细胞与人血隔绝;(四) MP 与中性粒细胞上清液 (由离心) 隔...

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本文介绍了中性粒细胞衍生 MPs 的分离和鉴定的协议。必须考虑到这一程序取得成功的几个关键关键点。首先, PMNs 必须隔离新鲜, 并在2小时内的隔离实验中使用, 以防止自发活化和脱粒。所有的 PMNs 在隔离和刺激点的处理必须在冰上执行, 以防止激活和过早 MP 释放12。第二, 离心为1小时或以上, 最大限度地提高了国会议员的造粒. 第三, 对于流式细胞术的分析, 必须用仪器指定的荧?...

最近, 来自细胞细胞质或等离子膜的 "微粒/微泡" 已成为极大的科学兴趣, 因为新兴的数据表明, 这些结构, 范围从 50-1, 000 nm 直径, 可以携带生物信息和作为一种非规范化的蜂窝通信方法。免疫细胞衍生的 MPs, 特别是中性粒细胞 (PMNs) 所产生的, 对于 PMNs 在宿主防御中的重要作用是很有兴趣的1,2, 炎症反应3, 伤口愈合4. 迄今为止, 许多报告都显示了中性粒细胞-mps5的亲炎症和抗炎作用, 表明了 mps 的潜在环境、疾病、物种和器官特定作用。
本通信中描述的协议为研究 MPs 在健康和疾病中的作用提供了一种成本效益高、创新性和适应性强的方法。它们适用于许多模型有机体, 器官和刺激条件。他们允许鉴定几种类型的 MPs, 并可用于未来, 以解决他们的亲炎和抗炎功能。例如, 这里描述了如何研究中性粒细胞-MPs 在上皮创面愈合中的作用体外和体内。提出了分离小鼠骨髓源性 PMNs 的协议, 并对先前描述的方法6进行了一些修改。
此外, 本研究中描述的协议可以通过两种互补的方法来检测和表征中性粒细胞 (MPs) 的特异标记物: 西方印迹和流式观察仪。我们发现使用标准协议5的 MPs 的免疫印迹法是容易和可靠的, 然而, 最近的进展, 流式细胞仪的灵敏度和改进的信噪比现在允许进一步分析 MPs 使用这种方法。本研究中所述的协议包括最近的研究成果和建议, 如对离心速度和时间的修改, 添加采样过滤和冻结/存储条件7,8, 以及如何减少 "背景噪声", 提高中性粒细胞的检测极限, 并区分不同大小的 mps。

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		<tr height="32">
			<td height="32" style="height:32px;width:257px;">DMEM</td>
			<td style="width:181px;">Corning</td>
			<td style="width:181px;">10-041-CV</td>
			<td style="width:348px;"></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:257px;">Fetal Bovine Serum (Heat inactivated)</td>
			<td style="width:181px;">Atlanta Biologics</td>
			<td style="width:181px;">S11150</td>
			<td style="width:348px;"></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:257px;">Penicillin/Streptomycin (10,000 units penicillin / 10,000 mg/ml strep)</td>
			<td style="width:181px;">GIBCO</td>
			<td style="width:181px;">15140</td>
			<td style="width:348px;"></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:257px;">0.5 M EDTA</td>
			<td style="width:181px;">Fisher</td>
			<td style="width:181px;">BP2482</td>
			<td style="width:348px;"></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:257px;">Sodium chloride</td>
			<td style="width:181px;">Sigma</td>
			<td style="width:181px;">S9888</td>
			<td style="width:348px;">Sterile and filtered through a 0.1 micron filter unit</td>
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		<tr height="32">
			<td height="32" style="height:32px;width:257px;">Sterile filtered Histopaque 1077</td>
			<td style="width:181px;">Sigma</td>
			<td style="width:181px;">10771</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:257px;">Sterile filtered Histopaque 1119</td>
			<td style="width:181px;">Sigma</td>
			<td style="width:181px;">11191</td>
			<td style="width:348px;">Density 1.119 g/ml.&#160; Solution of polysucrose and&#160; sodium diatrizoate. Bring to room temperature</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:257px;">Phosphate Buffered Saline (PBS) without Calcium and Magnesium</td>
			<td style="width:181px;">Cellgro</td>
			<td style="width:181px;">210-40-CV</td>
			<td style="width:348px;"></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:257px;">Ethyl Alcohol (200 proof)</td>
			<td style="width:181px;">Decon labs</td>
			<td style="width:181px;">2701</td>
			<td style="width:348px;"></td>
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		<tr height="32">
			<td height="32" style="height:32px;width:257px;">HBBS</td>
			<td style="width:181px;">Corning</td>
			<td style="width:181px;">21-022-CV</td>
			<td style="width:348px;"></td>
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		<tr height="32">
			<td height="32" style="height:32px;width:257px;">Trypan Blue Solution, 0.4%</td>
			<td style="width:181px;">Sigma</td>
			<td style="width:181px;">T8154&#160;SIGMA</td>
			<td style="width:348px;">Diluted 1:2 for cell viability counting</td>
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		<tr height="32">
			<td height="32" style="height:32px;width:257px;">Isoflurane</td>
			<td style="width:181px;">Abbot</td>
			<td style="width:181px;">50033</td>
			<td style="width:348px;"></td>
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		<tr height="32">
			<td height="32" style="height:32px;width:257px;">Anti-human CD11b-APC conjugated</td>
			<td style="width:181px;">Biolegend</td>
			<td style="width:181px;">301350</td>
			<td style="width:348px;">Clone ICRD44 (Final concetration: 1 &#181;g/mL)</td>
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		<tr height="32">
			<td height="32" style="height:32px;">BODIPY FL</td>
			<td>Invitrogen</td>
			<td>B10250</td>
			<td style="width:348px;">N-(2-aminoethyl) maleimide&#160;</td>
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		<tr height="32">
			<td height="32" style="height:32px;">Annexin V Binding Buffer</td>
			<td>Biolegend</td>
			<td>422201</td>
			<td style="width:348px;"></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">Calibration Beads for Flow Cytometry</td>
			<td>BioCytex</td>
			<td>7803</td>
			<td style="width:348px;"></td>
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		<tr height="32">
			<td height="32" style="height:32px;">FITC Annexin V</td>
			<td>Biolegend</td>
			<td>640906</td>
			<td style="width:348px;"></td>
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			<td height="32" style="height:32px;">Polymorphprep</td>
			<td>Axis-Shield PoC AS</td>
			<td></td>
			<td style="width:348px;">Sodium diatrizoate/Dextran 500, density 1.113 g/ml</td>
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		<tr height="18">
			<td height="18" style="height:19px;width:257px;">C57BL/6 mice</td>
			<td style="width:181px;">Jackson Labs</td>
			<td style="width:181px;"></td>
			<td style="width:348px;"></td>
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		<tr height="32">
			<td height="32" style="height:32px;width:257px;">15 ml centrifuge tubes</td>
			<td style="width:181px;">Corning</td>
			<td style="width:181px;">430053</td>
			<td style="width:348px;"></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:257px;">50 ml centrifuge tubes</td>
			<td style="width:181px;">BD Falcon</td>
			<td style="width:181px;">352070</td>
			<td style="width:348px;"></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:257px;">25 ml serological pipettes</td>
			<td style="width:181px;">Celltreat</td>
			<td style="width:181px;">229225B</td>
			<td style="width:348px;"></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:257px;">10 ml serological pipettes</td>
			<td style="width:181px;">Celltreat</td>
			<td style="width:181px;">229210B</td>
			<td style="width:348px;"></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:257px;">5 ml serological pipettes</td>
			<td style="width:181px;">Celltreat</td>
			<td style="width:181px;">229205B</td>
			<td style="width:348px;"></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:257px;">Pasteur pipettes&#160;</td>
			<td style="width:181px;">BD Falcon</td>
			<td style="width:181px;">357575</td>
			<td style="width:348px;"></td>
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		<tr height="18">
			<td height="18" style="height:19px;width:257px;">25 G x 5/8 in. Needles (precision glide needles)</td>
			<td style="width:181px;">BD</td>
			<td style="width:181px;">305122</td>
			<td style="width:348px;"></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:257px;">100 &#181;m cell strainers</td>
			<td style="width:181px;">Celltreat</td>
			<td style="width:181px;">229485</td>
			<td style="width:348px;"></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:257px;">Vacutainer tubes</td>
			<td style="width:181px;">BD</td>
			<td style="width:181px;">367251</td>
			<td style="width:348px;"></td>
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		<tr height="18">
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
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		<tr height="18">
			<td height="18" style="height:19px;">LSR Fortessa Special Order Research Product (SORP)</td>
			<td>BD</td>
			<td>(SORP)</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;">Swinging bucket centrifuge</td>
			<td>ThermoFisher Scientific</td>
			<td>75007210</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">Ultracentrifuge</td>
			<td>Beckman</td>
			<td>(L8-80 M)</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Micro-centrifuge</td>
			<td>ThermoFisher Scientific</td>
			<td>&#160;VV-17703-15 (Fresco 17)</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Swinging bucket centrifuge</td>
			<td>ThermoFisher Scientific</td>
			<td>&#160;75004503 (Megafuge 40R)</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Biopsy forceps, 28 cm</td>
			<td>Storz</td>
			<td>27071zj</td>
			<td></td>
		</tr>
		<tr height="17">
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Image J</td>
			<td>National Institute of Health</td>
			<td>Open source&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and characterization of neutrophil-derived microparticles for functional studies

中性粒细胞衍生微粒-MPs) 是脂双层, 球形微泡大小不等, 直径从 50–1,000 nm。MPs 是细胞对细胞通信和信号机械的一个新兴的、重要的组成部分。因为他们的大小和他们释放的性质, 直到最近 MP 存在被忽略了。然而, 随着技术和分析方法的改进, 它们在健康和疾病中的作用正在出现。这里提出的协议旨在通过流式细胞术和免疫印迹法分离和表征中性粒的 MPs。同时给出了几个实现实例。这些 MP 隔离协议是快速、低成本的, 不需要使用昂贵的套件。此外, 它们允许在隔离后对 MPs 进行标记, 以及在 MP 释放之前对源细胞进行预先标记, 使用膜特异荧光染料进行流式细胞仪的可视化和分析。然而, 这些方法有几个限制, 包括 PMNs 和 MPs 的纯度和复杂的分析仪器的需要。一个高端流式细胞仪是需要可靠地分析 MPs 和尽量减少假阳性读数由于噪声或自动荧光。所述协议可用于分离和定义 MP 合成, 并在不同刺激条件下表征其标记和组成的变化。可利用大小异质性研究膜颗粒与外来体的含量是否不同, 以及它们是否在组织稳态中发挥不同作用。最后, 在对 MPs 的分离和鉴定之后, 可以探讨它们在细胞反应和各种疾病模型 (包括炎症性肠病或急性肺损伤) 中的作用。

从人和鼠标 PMNs 隔离的 MPs 的代表性流细胞分析显示在图 1中。中性粒细胞的大小异质性可以通过与已知大小的珠子的比较来评估, 如图 1A、B中所示. 注意, 小鼠和人的 mps 在大小非均质上没有显著差异。同样, 使用流式细胞术和荧光标记, 可以确定由老鼠或人的孤立的 MPs 所选择的蛋白质/s 的表达。例如, 磷脂丝氨酸 (...

Watch this Scientific Journal Video about Isolation and Characterization of Neutrophil-derived Microparticles for Functional Studies at JoVE.com

Isolation and Characterization of Neutrophil-derived Microparticles for Functional Studies

本文介绍了新的协议, 以分离和表征从人和小鼠中性粒细胞衍生的微粒。这些协议利用离心、流式细胞术和免疫印迹法技术对微粒含量进行分析, 可用于研究不同细胞类型的微粒在细胞功能中的作用。

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Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

功能研究和过继转移实验小鼠骨髓嗜中性粒细胞的分离，纯化及贴标

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or ...

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

上皮损伤制作分析<em&gt;阿米巴</em&gt;感染

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

与碘酸雪夫染色检测糖原的外周血单个核细胞

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

Isolation and Characterization of Neutrophils with Anti-Tumor Properties

隔离和中性粒细胞的表征与抗肿瘤性质

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading ...

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

关于功能研究的三维人体肺组织模型<em&gt;结核分枝杆菌</em&gt;感染

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

分离，鉴定和牙龈免疫细胞网络的功能性考试

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

小鼠小肠淋巴细胞的分离和流式细胞表征

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...