Name: high throughput fluorometric technique for assessment of macrophage phagocytosis and actin polymerization
Uploaded: 2014-11-27T15:00:00
Description: Watch this Scientific Journal Video about High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization at JoVE.com

The authors would like to thank the following funding sources: RO1 DA 12104, RO1 DA 022935, RO1 DA031202, K05DA033881, P50 DA 011806, 1R01DA034582 (to S.R) and F31 DA026264-01A1, T32 DA07097 (to J.N.).

注意：此协议可以如在我们先前发表的作品12使用用于多种应用，如吞噬作用和肌动蛋白聚合的定量。的协议适合于各种变形例， 表1列出的细胞类型和颗粒在过去的研究中成功地使用。规范使用该协议的吞噬作用或肌动蛋白聚合的定量说明了图1。 <img alt="图1" fo:content-width="4in" src="/files/ftp_upload/52195/52195diagram1.jpg" w...

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所述荧光技术的主要限制是实验方差以及与广泛洗涤和使用非粘附细胞的细胞相关的损失。 方差很大程度上观察到由于在荧光发色粒子作为制备储液的过程中称重和分注的变化不保持从实验的颗粒进行实验的相同数量的精确方式。为了解决实验之间方差的问题，数据可以表示为相对术语，例如，作为％吞噬作用或折叠从控制变更（控制 - T = 0;加入和除去颗粒立即）。这种形?...

荧光信号的量化已被广泛应用于许多科学的方法，从PCR检测，流式细胞术，激光共聚焦显微镜和FRET分析复ELISA分析。荧光成像和定量有着广泛的应用，并且可针对各种细胞过程的定量分析的一大利器。使用的荧光标记物和它们的信号已经彻底改变了在过去十年中，并且荧光板阅读器的出现促进了荧光期间的细胞过程发出的光的高通量定量。 荧光分析可以作为吞噬量化一个伟大的工具。由于吞噬细胞的发现通过Metchnikoff在1800 1吞噬了研究。多年来，各种方法已被用于研究这个重要过程抵御入侵细菌，病毒，真菌和寄生虫病原体2-5先天免疫防御必需。量化利用显微镜和体视技术，以以前的方法以可视化的细胞被吞噬，然后将其通过计算内化颗粒（手动或使用利用软件）6-8的定量。一些缺点单独使用显微镜进行定量分析是细菌人工计数是劳动密集，更容易观察偏见。在吞噬的量化使用的另一种方法是从细胞裂解物（以下吞噬）上的细菌培养板细菌电镀的微生物技术，但这种方法可能会失败占杀菌机制和部分内在的细菌的存在。相比显微镜此方法甚至更多的劳动密集和需要几天的时间来分析。流式细胞术似乎是量化吞噬的最快，最有效的方法，并已用于通过许多组9-11，但成本高，通常与在相关联的所需的分析仪器使得它的最昂贵的方法时，相比于前面提到的测定。 该荧光法是一个很好的替代流式细胞仪粒子内部的分析，因为它提供了用荧光设备偏见的量化，是不是因为成本太高。荧光的其它额外的好处是效率高，高吞吐量能力用于定量荧光的标记的内化的颗粒排出。 荧光的好处是可以外推到比吞噬其它过程定量。例如，荧光分析，可用于研究任何导致肌动蛋白聚合的变化在细胞内或膜结合的受体，改变细胞通透性/生存力，转染效率，以及调制的表达过程。荧光技术的一个缺点是，根据所使用的标签，可能有一个高实验实验变异性可通常通过证明通过相对定量数据，如倍数变化或从控制增加的百分比来解决。

<table border="0" cellpadding="0" cellspacing="0" width="820">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Name of Material/ Equipment</td>
			<td style="width:131px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:255px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;"></td>
			<td style="width:131px;"></td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="66">
			<td height="66" style="height:66px;width:315px;">1-&#956;m yellow-green fluorescent Fluo-Spheres;</td>
			<td style="width:131px;">Molecular Probes</td>
			<td style="width:119px;">F8852</td>
			<td style="width:255px;">combine 50&#956;l with 50&#956;l opsonizing reagent for 30 min at 37oC before use</td>
		</tr>
		<tr height="87">
			<td height="87" style="height:87px;">Heat killed E. coli BioParticles fluorescein conjugate</td>
			<td style="width:131px;">Molecular Probes</td>
			<td style="width:119px;">E2861</td>
			<td style="width:255px;">reconstitute (5 mg) in 50&#956;l of PBS and combine with 50&#956;l opsonizing reagent for 30 min at 37oC before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Opsonizing reagent</td>
			<td style="width:131px;">Molecular Probes</td>
			<td style="width:119px;">E2870</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Rhodamine phalloidin</td>
			<td style="width:131px;">Molecular Probes</td>
			<td style="width:119px;">R415</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Trypan blue</td>
			<td style="width:131px;">Gibco</td>
			<td style="width:119px;">15250-061</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;"></td>
			<td style="width:131px;"></td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;"></td>
			<td style="width:131px;"></td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;">the items below are available in many brands but the items we used in this study are from the following manufacturers</td>
		</tr>
		<tr height="66">
			<td height="66" style="height:66px;width:315px;">RPMI Cell growth media&#160;</td>
			<td style="width:131px;">Gibco</td>
			<td style="width:119px;"></td>
			<td style="width:255px;">Supplemented with 10% FBS and 1% pennicillin/Streptomycin; warm in 37oC before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Fluorescent plate reader-Fluostar Omega</td>
			<td style="width:131px;">BMG Labtech</td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Paraformaldehyde (16%)</td>
			<td style="width:131px;">Fischer Scientific</td>
			<td>AA433689M</td>
			<td style="width:255px;">dilute to 4% before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">96 well plates</td>
			<td style="width:131px;">Greiner</td>
			<td style="width:119px;">655097</td>
			<td style="width:255px;">clear or black or clear bottom - black plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Multichannel pipette (8-12 channels)</td>
			<td style="width:131px;"></td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Reagent reservoirs</td>
			<td style="width:131px;"></td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">1x PBS</td>
			<td style="width:131px;"></td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Microfuge tubes (0.6 ml)</td>
			<td style="width:131px;"></td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">&#160;Conicles (10 ml)</td>
			<td style="width:131px;"></td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
	</tbody>
</table>

high throughput fluorometric technique for assessment of macrophage phagocytosis and actin polymerization

荧光分析的目的是作为一种有效的，成本有效的，高通量分析的吞噬作用和其他细胞过程的方法。这种技术可以用在各种细胞类型，包括粘附和非粘附的，以检查各种细胞特性。当研究吞噬作用，荧光技术利用吞噬细胞类型如巨噬细胞，和荧光标记的调理粒子的荧光可以以台盼蓝的存在而熄灭。下面粘附的巨噬细胞的电镀在96孔板中，荧光发色粒子（绿或红）施用和允许细胞吞噬对于不同量的时间。以下荧光体颗粒的内在化，细胞用台盼蓝，这有利于荧光信号的消光从其中不内化，或仅仅是附着于细胞表面的细菌。继锥虫洗涤，将细胞用PBS洗涤，固定，一次用DAPI染色（蓝色核荧光标记），其用于标记细胞的细胞核。通过板的核（蓝）读取或颗粒的简单荧光定量（红/绿）荧光我们可以检查的绿色相对荧光单位的比例：蓝色和确定一个表示每个细胞内在荧光的细菌量的吞噬指数。测定使用96孔的方法和多道移液器用于洗涤的持续时间，从吞噬数据采集结束的结束，是小于45分钟。流式细胞术以类似的方式也可以使用，但荧光的优点是它的高通量，评价与样品最小操纵和每个细胞的荧光强度的快速定量快速方法。类似的策略可以应用到非粘附细胞，活标记的细菌，肌动蛋白聚合，并且基本上任何利用荧光过程。因此，荧光是一种有前途的方法，其成本低，高throughpUT能力的细胞过程的研究。

通过使用该协议的量化吞噬作用和随后的肌动蛋白聚合的两种主要方法是观察连续或同步吞噬作用。 显微镜分析（ 图1B）示出了荧光图像媲美什么荧光正在记录（也参见图1）。在图1B是红色荧光的肌动蛋白，绿色荧光268 FITC的标签HKOP E.大肠杆菌 ，核DAPI染色，三个人269的合并后的图像荧光团在一起。这个图像显示有效的吞噬作用，...

Watch this Scientific Journal Video about High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization at JoVE.com

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.

高通量荧光技术巨噬细胞吞噬功能和肌动蛋白聚合的评估

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular ...

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