作者要感谢微生物学，免疫学和生物化学系高级插图师Tim Higgins先生提供技术视频协助; Matthew W. Wilson博士进行讨论，还有Jablonski和Morales-Tirado实验室的成员对他们的帮助意见。这项工作得到了阿尔康研究院青年研究员奖（VMM-T），田纳西大学研究基金会（VMM-T），国家眼科学院EY021200（MMJ），格温奖学金（VMM-T）等的支持。格温博士后研究金（ZKG），国防军医学研究和物资司令部（VMM-T）以及研究预防盲人无限制赠款。

<ol>
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作者宣称他们没有竞争的经济利益。

FACS是净化细胞群体的首选技术。其他分离方法包括免疫沉淀，磁珠和补体固定消耗。 FACS与这些其他方法的优点是基于同时鉴别具有不同程度的强度的细胞表面标志物。分子的荧光强度与蛋白质表达量成比例。到目前为止，RGC的分离仅基于Thy1（CD90）阳性和CD48阴性15,16,22,34，而不管使用的分离方法如何。最近已经显示，Thy1 + CD48 neg表型不足以分离表达RGC细胞内标志物...

RGC是终末分化的神经元，因此，原代细胞是实验所必需的。用于分离和富集原代小鼠视网膜神经节细胞（RGC）的方案的开发是揭示RGC健康和体外变性的机制的基础。这对于寻求产生潜在疗法以促进RGC功能并尽量减少死亡率的研究尤为重要。 RGC的变性与视网膜退行性疾病如青光眼，糖尿病性视网膜病变和正常衰老有关。虽然RGC损失的具体细胞机制尚不清楚，但已经确定了一系列危险因素。缺乏视神经头1,2,3的氧合导致RGC死亡4 ，并且作为兴奋性和i激活之间的动态平衡的干扰个体RGCs内的抑制性受体5,6 。一系列挑战妨碍了使用这些细胞进行深入研究的进展。首先，鼠视网膜中RGCs的数量很少。 RGC占总视网膜细胞7,8,9的不到1％。第二，大多数RGC特异性标记物是细胞内蛋白质10,11,12 。基于这些标记的选择使细胞不可行，这排除了下游功能分析。最后，目前可用的协议冗长，缺乏标准化13,14 。早期RGC分离方案是基于免疫方法。 Barres 等人 15 适应经典免疫基于对抗胸腺细胞抗原（也称Thy1）（细胞表面标记）的免疫阳性，在阳性选择之前，将大部分视网膜细胞从单核细胞和内皮细胞排除第二步。几年后，Hong et al。组合磁珠分离技术与细胞分选策略分离更高纯度的RGC 16 。在许多科学应用中仍然使用磁珠。一起，磁珠和流式细胞术方案提高了分离细胞的纯度。然而，这些纯化系统尚未被标准化用于从解离的视网膜分离鼠RGCs。 流式细胞仪是一种强大的分析方法，可以测量细胞悬浮液的光学和荧光特性。以高水平的灵敏度定量和定性分析细胞，为细胞群体提供多维分析通货膨胀。细胞鉴别基于两个主要物理性质:细胞大小或表面积，粒度或内部复杂性17 。可以通过组合具有类似激发波长和不同排放物的荧光染料标记的抗体来进行多维分析。流式细胞术是快速，可重复和敏感的。多重激光器通过流式细胞术允许单个细胞的更多的多维分析。因此，它是研究细胞学标本的有吸引力的方法。荧光激活细胞分选（FACS）使用通过流式细胞术鉴定的多维表型差异将单个细胞分类为不同的亚群。 在过去十年中，多种表面和细胞内蛋白已被鉴定为用于选择细胞（包括神经元）的潜在生物标志物。试图从大鼠中分离RGC的初步研究使用Thy1作为神经节细胞l标记。不幸的是，Thy1， 也就是 CD90，在其他啮齿动物物种18,19,20中具有多个同种型，并且由多个视网膜细胞类型19,20表达，使其成为RGC的非特异性标记。在视网膜中的单核细胞群体中发现另一个表面标志物CD48，包括巨噬细胞和小神经胶质细胞。使用这两个表面标记，修饰的RGC特征-Thy1 +和CD48 neg细胞 - 被开发出15,16,21,22。不幸的是，这两个选择标准不足以选择高度富集的研资局人口。为了解决这一未满足的需求，基于多层次正，负选择标准，开发了流式细胞仪方案23已知的细胞表面标志物以富集和纯化原代鼠RGC。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anti-mouse CD15 PE</td>
			<td>BioLegend</td>
			<td>125606</td>
			<td>Clone MC-480</td>
		</tr>
		<tr>
			<td>Anti-mouse CD48 PE-Cy7</td>
			<td>BioLegend</td>
			<td>103424</td>
			<td>Clone HM48-1</td>
		</tr>
		<tr>
			<td>Anti-mouse CD57</td>
			<td>Sigma Aldrich</td>
			<td>C6680-100TST</td>
			<td>Clone VC1.1</td>
		</tr>
		<tr>
			<td>Anti-mouse CD90.2 AF700</td>
			<td>BioLegend</td>
			<td>105320</td>
			<td>Clone 30-H12</td>
		</tr>
		<tr>
			<td>Brilliant Violet 421 Goat Anti-mouse IgG</td>
			<td>BioLegend</td>
			<td>405317</td>
			<td>Clone Poly4053</td>
		</tr>
		<tr>
			<td>Purified Anti-mouse CD16/32</td>
			<td>BioLegend</td>
			<td>101302</td>
			<td>FcgRII/III block, Clone 93</td>
		</tr>
		<tr>
			<td>Zombie Aqua&#160;</td>
			<td>BioLegend</td>
			<td>423102</td>
			<td>Live cell/ Dead cell discrimination</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Hyclone</td>
			<td>SH30071.03</td>
			<td>U.S. origin</td>
		</tr>
		<tr>
			<td>AbC Total Antibody Compensation Bead Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>A10497</td>
			<td>Multi-species Ig</td>
		</tr>
		<tr>
			<td>Neurobasal Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>21103049</td>
			<td>Add serum to media prior to culture.</td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>10010049</td>
			<td>Saline solution</td>
		</tr>
		<tr>
			<td>Dissection Microscope</td>
			<td>Olympus</td>
			<td>SZ-PT Model</td>
			<td>Stereo Microscope</td>
		</tr>
		<tr>
			<td>Sorvall Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>ST 16R</td>
			<td>All centrifugation performed&#160; at RT</td>
		</tr>
		<tr>
			<td>Base Plate &#8211; Dissection Pan</td>
			<td>Fisher Scientific</td>
			<td>SB15233FIM</td>
			<td>A wax plate can also be used</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Aesculap</td>
			<td>5002-7</td>
			<td>4 &#189; inches</td>
		</tr>
		<tr>
			<td>Iris Scissors, Straight</td>
			<td>Aesculap</td>
			<td>1360</td>
			<td>5 &#189; inches</td>
		</tr>
		<tr>
			<td>Falcon 15 mL conical tubes</td>
			<td>Fisher Scientific</td>
			<td>352097</td>
			<td>Polypropylene tubes</td>
		</tr>
		<tr>
			<td>Falcon 50 mL conical tubes</td>
			<td>Fisher Scientific</td>
			<td>352098</td>
			<td>Polypropylene tubes</td>
		</tr>
		<tr>
			<td>BD FACS Tubes</td>
			<td>Fisher Scientific</td>
			<td>352003</td>
			<td>Polypropylene tubes</td>
		</tr>
		<tr>
			<td>40 mm dishes</td>
			<td>MidSci</td>
			<td>TP93040</td>
			<td>Tissue culture treated</td>
		</tr>
		<tr>
			<td>70 &#956;m nylon strainer</td>
			<td>MidSci</td>
			<td>70ICS</td>
			<td>sterile</td>
		</tr>
		<tr>
			<td>40 &#956;m nylon strainer</td>
			<td>MidSci</td>
			<td>40ICS</td>
			<td>sterile</td>
		</tr>
		<tr>
			<td>&#160;BD 10 mL syringe</td>
			<td>Fisher Scientific</td>
			<td>301604</td>
			<td>Disposable Syringe without needle</td>
		</tr>
		<tr>
			<td>Pestles</td>
			<td>MidSci</td>
			<td>PEST</td>
			<td>sterile</td>
		</tr>
		<tr>
			<td>Wheaton Vials</td>
			<td>Fisher Scientific</td>
			<td>986734</td>
			<td>No Liner</td>
		</tr>
		<tr>
			<td>BD 30 G needle</td>
			<td>Fisher Scientific</td>
			<td>305128</td>
			<td>1 inch</td>
		</tr>
		<tr>
			<td>Hausser Scientific Bright-Line Glass Counting Chamber</td>
			<td>Fisher Scientific</td>
			<td>0267151B</td>
			<td>Hemocytometer</td>
		</tr>
		<tr>
			<td>Gibco Trypan blue 0.4% Solution</td>
			<td>Fisher Scientific</td>
			<td>15250061</td>
			<td>Viability Dye</td>
		</tr>
		<tr>
			<td>Eppendorf tubes</td>
			<td>Fisher Scientific</td>
			<td>05-402-25</td>
			<td>1.5mL</td>
		</tr>
		<tr>
			<td>EVOS Floid Cell Imaging</td>
			<td>Thermo Fisher Scientific</td>
			<td>447113</td>
			<td>Fluorescence Imaging with a 20x objective</td>
		</tr>
		<tr>
			<td>100% Ethanol</td>
			<td>Fisher Scientific</td>
			<td>04-355-452</td>
			<td>Used to make 70% Ethanol</td>
		</tr>
		<tr>
			<td>Pipet-Lite LTS Pipette L-1000XLS+</td>
			<td>Rainin</td>
			<td>17014282</td>
			<td>LTS Pipette</td>
		</tr>
		<tr>
			<td>Pipet-Lite LTS Pipette L-200XLS+</td>
			<td>Rainin</td>
			<td>17014391</td>
			<td>LTS Pipette</td>
		</tr>
		<tr>
			<td>Pipet-Lite LTS Pipette L-20XLS+</td>
			<td>Rainin</td>
			<td>17014392</td>
			<td>LTS Pipette</td>
		</tr>
		<tr>
			<td>Rack LTS 1000 mL &#8211; GPS-L1000S</td>
			<td>Rainin</td>
			<td>17005088</td>
			<td>Blue Rack Sterile Tips</td>
		</tr>
		<tr>
			<td>Rack LTS 250 mL &#8211; GPS-L250S</td>
			<td>Rainin</td>
			<td>17005092</td>
			<td>Green Rack Sterile Tips</td>
		</tr>
		<tr>
			<td>Rack LTS 20 mL &#8211; GPS-L10S</td>
			<td>Rainin</td>
			<td>17005090</td>
			<td>Red Rack Sterile Tips</td>
		</tr>
		<tr>
			<td>FACSAria II Cell Sorter</td>
			<td>BD Biosciences</td>
			<td>N/A</td>
			<td>Custom order</td>
		</tr>
		<tr>
			<td>LSR II Cytometer</td>
			<td>BD Biosciences</td>
			<td>N/A</td>
			<td>Custom order</td>
		</tr>
		<tr>
			<td>Abca8a</td>
			<td>Thermo Fisher Scientific</td>
			<td>Mm00462440_m1</td>
			<td>M&#252;ller cells</td>
		</tr>
		<tr>
			<td>Aldh1al</td>
			<td>Thermo Fisher Scientific</td>
			<td>Mm00657317_m1</td>
			<td>M&#252;ller cells</td>
		</tr>
		<tr>
			<td>Aqp4</td>
			<td>Thermo Fisher Scientific</td>
			<td>Mm00802131_m1</td>
			<td>Astrocytes</td>
		</tr>
		<tr>
			<td>Calb2</td>
			<td>Thermo Fisher Scientific</td>
			<td>Mm00801461_m1</td>
			<td>Amacrine, Horizontal</td>
		</tr>
		<tr>
			<td>Cd68</td>
			<td>Thermo Fisher Scientific</td>
			<td>Mm03047340_m1</td>
			<td>Retinal Pigment Epithelial Cells</td>
		</tr>
		<tr>
			<td>Gad2</td>
			<td>Thermo Fisher Scientific</td>
			<td>Mm00484623_m1</td>
			<td>Amacrine</td>
		</tr>
		<tr>
			<td>Hprt</td>
			<td>Thermo Fisher Scientific</td>
			<td>Mm01545399_m1</td>
			<td>House keeping gene</td>
		</tr>
		<tr>
			<td>Lhx1</td>
			<td>Thermo Fisher Scientific</td>
			<td>Mm01297482_m1</td>
			<td>Horizontal</td>
		</tr>
		<tr>
			<td>Lim2</td>
			<td>Thermo Fisher Scientific</td>
			<td>Mm00624623_m1</td>
			<td>Horizontal</td>
		</tr>
		<tr>
			<td>Nrl</td>
			<td>Thermo Fisher Scientific</td>
			<td>Mm00476550_m1</td>
			<td>Photoreceptors</td>
		</tr>
		<tr>
			<td>Ntrk1</td>
			<td>Thermo Fisher Scientific</td>
			<td>Mm01219406_m1</td>
			<td>Horizontal</td>
		</tr>
		<tr>
			<td>Pcp4</td>
			<td>Thermo Fisher Scientific</td>
			<td>Mm00500973_m1</td>
			<td>Bipolar, Amacrine</td>
		</tr>
		<tr>
			<td>Pou4f1</td>
			<td>Thermo Fisher Scientific</td>
			<td>Mm02343791_m1</td>
			<td>Retinal Ganglion Cells</td>
		</tr>
		<tr>
			<td>Prdx6</td>
			<td>Thermo Fisher Scientific</td>
			<td>Mm00725435_s1</td>
			<td>Astrocytes</td>
		</tr>
		<tr>
			<td>Prkca</td>
			<td>Thermo Fisher Scientific</td>
			<td>Mm00440858_m1</td>
			<td>Bipolar</td>
		</tr>
		<tr>
			<td>Prox1</td>
			<td>Thermo Fisher Scientific</td>
			<td>Mm00435969_m1</td>
			<td>Horizontal</td>
		</tr>
		<tr>
			<td>Pvalb</td>
			<td>Thermo Fisher Scientific</td>
			<td>Mm00443100_m1</td>
			<td>Amacrine</td>
		</tr>
		<tr>
			<td>Rbpms</td>
			<td>Thermo Fisher Scientific</td>
			<td>Mm02343791_m1</td>
			<td>Retinal Ganglion Cells</td>
		</tr>
		<tr>
			<td>Rom1</td>
			<td>Thermo Fisher Scientific</td>
			<td>Mm00436364_g1</td>
			<td>Photoreceptors</td>
		</tr>
		<tr>
			<td>Rpe65</td>
			<td>Thermo Fisher Scientific</td>
			<td>Mm00504133_m1</td>
			<td>Retinal Pigment Epithelial cells</td>
		</tr>
		<tr>
			<td>Slc1a3</td>
			<td>Thermo Fisher Scientific</td>
			<td>Mm00600697_m1</td>
			<td>Astrocytes</td>
		</tr>
		<tr>
			<td>Slc6a9</td>
			<td>Thermo Fisher Scientific</td>
			<td>Mm00433662_m1</td>
			<td>Amacrine</td>
		</tr>
		<tr>
			<td>Sncg</td>
			<td>Thermo Fisher Scientific</td>
			<td>Mm00488345_m1</td>
			<td>Retinal Ganglion Cells</td>
		</tr>
		<tr>
			<td>Tubb3</td>
			<td>Thermo Fisher Scientific</td>
			<td>Mm00727586_s1</td>
			<td>Retinal Ganglion Cells</td>
		</tr>
		<tr>
			<td>Vim</td>
			<td>Thermo Fisher Scientific</td>
			<td>Mm01333430_m1</td>
			<td>M&#252;ller cells</td>
		</tr>
		<tr>
			<td>Taqman Universal Master Mix</td>
			<td>Thermo Fisher Scientific</td>
			<td>4440047</td>
			<td>qPCR Reagent</td>
		</tr>
		<tr>
			<td>miRNeasy Mini Kit</td>
			<td>Qiagen</td>
			<td>217004</td>
			<td>RNA Isolation</td>
		</tr>
		<tr>
			<td>SuperScript VILO cDNA Synthesis Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>11754250</td>
			<td>cDNA synthesis</td>
		</tr>
		<tr>
			<td>Taqman PreAmp Master Mix</td>
			<td>Thermo Fisher Scientific</td>
			<td>4391128</td>
			<td>Pre-Amplification step</td>
		</tr>
		<tr>
			<td>BD Cytofix/ Cytoperm</td>
			<td>BD Biosciences</td>
			<td>554714</td>
			<td>Fixation/ Permeabilization Buffer</td>
		</tr>
		<tr>
			<td>BD Perm/ Wash</td>
			<td>BD Biosciences</td>
			<td>554723</td>
			<td>Permeabilization Solution</td>
		</tr>
		<tr>
			<td>RBPMS</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-86815</td>
			<td>intracellular antibody</td>
		</tr>
		<tr>
			<td>SNCG</td>
			<td>Gene Tex</td>
			<td>GTX110483</td>
			<td>intracellular antibody</td>
		</tr>
		<tr>
			<td>BRN3A</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-8429</td>
			<td>intracellular antibody</td>
		</tr>
		<tr>
			<td>TUJ1</td>
			<td>BioLegend</td>
			<td>801202</td>
			<td>intracellular antibody</td>
		</tr>
	</tbody>
</table>

isolation of primary murine retinal ganglion cells (rgcs) by flow cytometry

神经退行性疾病往往对受影响者有破坏性的影响。除了正常老化之外，视网膜神经节细胞（RGC）损失也涉及一系列疾病，包括糖尿病性视网膜病变和青光眼。尽管它们的重要性，RGC迄今为止非常难以研究，部分原因在于它们仅占视网膜中各种细胞的一小部分。此外，目前的分离方法使用细胞内标记来鉴定产生非活细胞的RGC。这些技术还涉及冗长的分离方案，因此缺乏获得和分离RGC的实用，标准化和可靠的方法。这项工作描述了一种有效，全面和可靠的方法，使用基于正选择标准和阴性选择标准的方案从小鼠视网膜分离原代RGC。所提出的方法允许未来RGCs的研究，目的是更好地了解专业由神经退行性疾病中功能性RGCs的丧失导致的视力下降。

<html> RGC的深入研究受到许多因素的阻碍，包括其频率低，缺乏强大和标准化的隔离方法。 图1显示了用于视网膜分离的方法。根据分析方法存在摘除程序的变化，例如摘除核素是体内实验的一部分27 。在安乐死小鼠中进行该方案的摘除。 如图1A- B所示，镊子被放置在眼睛下方并被拉起以引?...

Watch this Scientific Journal Video about Isolation of Primary Murine Retinal Ganglion Cells RGCs by Flow Cytometry at JoVE.com

Isolation of Primary Murine Retinal Ganglion Cells RGCs by Flow Cytometry

数百万人患有导致不可逆失明的视网膜退行性疾病。许多这些疾病的常见因素是视网膜神经节细胞（RGC）的丧失。该详细方案描述了通过流式细胞术的阳性和阴性选择分离原代鼠RGC。

通过流式细胞术分离原代小鼠视网膜神经节细胞（RGC）

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, ...

isolation-primary-murine-retinal-ganglion-cells-rgcs-flow

Research

JoVE Journal

Bioengineering

Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry

14.8K 次观看.  University of Tennessee Health Science Center. 数百万人患有导致不可逆失明的视网膜退行性疾病。许多这些疾病的常见因素是视网膜神经节细胞（RGC）的丧失。该详细方案描述了通过流式细胞术的阳性和阴性选择分离原代鼠RGC。 

视频: Isolation of Primary Murine Retinal Ganglion Cells RGCs by Flow Cytometry

Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well quantifiable fluid shear stresses1.
Our flow chamber design and flow circuit (Fig. 1) contains a transparent viewing region that enables testing of cell adhesion and imaging of cell morphology immediately before flow (Fig. 11A, B), at various time points during flow (Fig. 11C), and after flow (Fig. 11D). These experiments are illustrated with human umbilical cord blood-derived endothelial progenitor cells (EPCs) and porcine EPCs2,3.
This method is also applicable to other adherent cell types, e.g. smooth muscle cells (SMCs) or fibroblasts.
The chamber and all parts of the circuit are easily sterilized with steam autoclaving. In contrast to other chambers, e.g. microfluidic chambers, large numbers of cells (&gt; 1 million depending on cell size) can be recovered after the flow experiment under sterile conditions for cell culture or other experiments, e.g. DNA or RNA extraction, or immunohistochemistry (Fig. 11E), or scanning electron microscopy5. The shear stress can be adjusted by varying the flow rate of the perfusate, the fluid viscosity, or the channel height and width. The latter can reduce fluid volume or cell needs while ensuring that one-dimensional flow is maintained. It is not necessary to measure chamber height between experiments, since the chamber height does not depend on the use of gaskets, which greatly increases the ease of multiple experiments. Furthermore, the circuit design easily enables the collection of perfusate samples for analysis and/or quantification of metabolites secreted by cells under fluid shear stress exposure, e.g. nitric oxide (Fig. 12)6.

We are describing a method to subject adherent cells to laminar flow shear stress in a sterile continuous flow circuit. The cells' adhesion, morphology can be studied through the transparent chamber, samples obtained from the circuit for metabolite analysis and cells harvested after shear exposure for future experiments or culture.

平行板流动腔和评估内皮祖细胞层流剪应力下连续流电路

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well ...

科研

生物工程

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

Nanoparticulate systems have emerged as valuable tools in vaccine delivery through their ability to efficiently deliver cargo, including proteins, to antigen presenting cells1-5. Internalization of nanoparticles (NP) by antigen presenting cells is a critical step in generating an effective immune response to the encapsulated antigen. To determine how changes in nanoparticle formulation impact function, we sought to develop a high throughput, quantitative experimental protocol that was compatible with detecting internalized nanoparticles as well as bacteria. To date, two independent techniques, microscopy and flow cytometry, have been the methods used to study the phagocytosis of nanoparticles. The high throughput nature of flow cytometry generates robust statistical data. However, due to low resolution, it fails to accurately quantify internalized versus cell bound nanoparticles. Microscopy generates images with high spatial resolution; however, it is time consuming and involves small sample sizes6-8. Multi-spectral imaging flow cytometry (MIFC) is a new technology that incorporates aspects of both microscopy and flow cytometry that performs multi-color spectral fluorescence and bright field imaging simultaneously through a laminar core. This capability provides an accurate analysis of fluorescent signal intensities and spatial relationships between different structures and cellular features at high speed.
	Herein, we describe a method utilizing MIFC to characterize the cell populations that have internalized polyanhydride nanoparticles or Salmonella enterica serovar Typhimurium. We also describe the preparation of nanoparticle suspensions, cell labeling, acquisition on an ImageStreamX system and analysis of the data using the IDEAS application. We also demonstrate the application of a technique that can be used to differentiate the internalization pathways for nanoparticles and bacteria by using cytochalasin-D as an inhibitor of actin-mediated phagocytosis.

In this article, we describe a method utilizing multi-spectral imaging flow cytometry to quantify the internalization of polyanhydride nanoparticles or bacteria by RAW 264.7 cells.

由多光谱成像流式细胞仪分析细胞内化纳米粒子和细菌

Nanoparticulate  systems have emerged as valuable tools in vaccine delivery through their  ability to efficiently deliver cargo, including proteins, to ...

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

高分子基因传递纳米粒子的纳米粒子跟踪分析和高通量流式细胞仪评价

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Wide-field Fluorescent Microscopy and Fluorescent Imaging Flow Cytometry on a Cell-phone

Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general relatively bulky and costly, making them less effective in the resource limited settings. To potentially address these limitations, we have recently demonstrated the integration of wide-field fluorescent microscopy and imaging flow cytometry tools on cell-phones using compact, light-weight, and cost-effective opto-fluidic attachments. In our flow cytometry design, fluorescently labeled cells are flushed through a microfluidic channel that is positioned above the existing cell-phone camera unit. Battery powered light-emitting diodes (LEDs) are butt-coupled to the side of this microfluidic chip, which effectively acts as a multi-mode slab waveguide, where the excitation light is guided to uniformly excite the fluorescent targets. The cell-phone camera records a time lapse movie of the fluorescent cells flowing through the microfluidic channel, where the digital frames of this movie are processed to count the number of the labeled cells within the target solution of interest. Using a similar opto-fluidic design, we can also image these fluorescently labeled cells in static mode by e.g. sandwiching the fluorescent particles between two glass slides and capturing their fluorescent images using the cell-phone camera, which can achieve a spatial resolution of e.g. ~ 10 &mu;m over a very large field-of-view of ~ 81 mm2. This cell-phone based fluorescent imaging flow cytometry and microscopy platform might be useful especially in resource limited settings, for e.g. counting of CD4+ T cells toward monitoring of HIV+ patients or for detection of water-borne parasites in drinking water.

We review our recent results on the integration of fluorescent microscopy and imaging flow cytometry tools on a cell-phone using compact and cost-effective opto-fluidic attachments. These cell-phone based micro-analysis devices might be useful for cytometric analysis, such as performing various cell counting tasks as well as for high-throughput screening of e.g., water samples in resource limited settings.

一个手机上的宽视场荧光显微镜和荧光成像流式细胞仪

Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general ...

Localization and Relative Quantification of Carbon Nanotubes in Cells with Multispectral Imaging Flow Cytometry

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich cell structures and de facto there is still no quantitative method to assess their distribution at cell and tissue levels. What we propose here is an innovative method allowing the detection and quantification of CNTs in cells using a multispectral imaging flow cytometer (ImageStream, Amnis). This newly developed device integrates both a high-throughput of cells and high resolution imaging, providing thus images for each cell directly in flow and therefore statistically relevant image analysis. Each cell image is acquired on bright-field (BF), dark-field (DF), and fluorescent channels, giving access respectively to the level and the distribution of light absorption, light scattered and fluorescence for each cell. The analysis consists then in a pixel-by-pixel comparison of each image, of the 7,000-10,000 cells acquired for each condition of the experiment. Localization and quantification of CNTs is made possible thanks to some particular intrinsic properties of CNTs: strong light absorbance and scattering; indeed CNTs appear as strongly absorbed dark spots on BF and bright spots on DF with a precise colocalization.
This methodology could have a considerable impact on studies about interactions between nanomaterials and cells given that this protocol is applicable for a large range of nanomaterials, insofar as they are capable of absorbing (and/or scattering) strongly enough the light.

In this study, the main purpose was to monitor the cellular uptake and eventual exocytosis of carbon nanotubes (CNTs). From this perspective, we proposed here a unique method using multispectral imaging flow cytometry allowing a quantification and localization of CNTs in a statistically relevant number of cells.

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich ...

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion receptors and associated signal transduction membrane proteins, the cytoskeletal architecture, and molecular motors1, 2. Mechanosensitivity of different cancer cells in vitro are investigated primarily with immortalized cell lines or murine derived primary cells, not with primary human cancer cells. Hence, little is known about the mechanosensitivity of primary human colon cancer cells in vitro. Here, an optimized protocol is developed that describes the isolation of primary human colon cells from healthy and cancerous surgical human tissue samples. Isolated colon cells are then successfully cultured on soft (2 kPa stiffness) and stiff (10 kPa stiffness) polyacrylamide hydrogels and rigid polystyrene (~3.6 GPa stiffness) substrates functionalized by an extracellular matrix (fibronectin in this case). Fluorescent microbeads are embedded in soft gels near the cell culture surface, and traction assay is performed to assess cellular contractile stresses using free open access software. In addition, immunofluorescence microscopy on different stiffness substrates provides useful information about primary cell morphology, cytoskeleton organization and vinculin containing focal adhesions as a function of substrate rigidity.

A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses.

从手术组织小学的人结肠癌细胞的分离和培养直接它们柔软的弹性基底牵引术

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion ...

Sensitive Detection of Proteopathic Seeding Activity with FRET Flow Cytometry

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and progression of pathology in several neurodegenerative diseases, including Alzheimer's disease and the related tauopathies. The potentially critical role of tau seeds in disease progression strongly supports the need for a sensitive assay that readily detects seeding activity in biological samples. 
By combining the specificity of fluorescence resonance energy transfer (FRET), the sensitivity of flow cytometry, and the stability of a monoclonal cell line, an ultra-sensitive seeding assay has been engineered and is compatible with seed detection from recombinant or biological samples, including human and mouse brain homogenates. The assay employs monoclonal HEK 293T cells that stably express the aggregation-prone repeat domain (RD) of tau harboring the disease-associated P301S mutation fused to either CFP or YFP, which produce a FRET signal upon protein aggregation. The uptake of proteopathic tau seeds (but not other proteins) into the biosensor cells stimulates aggregation of RD-CFP and RD-YFP, and flow cytometry sensitively and quantitatively monitors this aggregation-induced FRET. The assay detects femtomolar concentrations (monomer equivalent) of recombinant tau seeds, has a dynamic range spanning three orders of magnitude, and is compatible with brain homogenates from tauopathy transgenic mice and human tauopathy subjects. With slight modifications, the assay can also detect seeding activity of other proteopathic seeds, such as &#945;-synuclein, and is also compatible with primary neuronal cultures. The ease, sensitivity, and broad applicability of FRET flow cytometry makes it useful to study a wide range of protein aggregation disorders.

Cell-to-cell transfer of protein aggregates, or proteopathic seeds, may underlie the progression of pathology in neurodegenerative diseases. Here, a novel FRET flow cytometry assay is described that enables specific and sensitive detection of seeding activity from recombinant or biological samples.

与FRET流式细胞仪灵敏地检测Proteopathic播种活动

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and ...

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of (indirect) in vitro experiments, but none of these is as comprehensive as microfluidic flow chambers. In this protocol, the reconstitution of thrombocytopenic fresh blood with stored blood bank platelets is used to simulate platelet transfusion. Next, the reconstituted sample is perfused in microfluidic flow chambers which mimic hemostasis on exposed subendothelial matrix proteins. Effects of blood donation, transport, component separation, storage and pathogen inactivation can be measured in paired experimental designs. This allows reliable comparison of the impact every manipulation in blood component preparation has on hemostasis. Our results demonstrate the impact of temperature cycling, shear rates, platelet concentration and storage duration on platelet function. In conclusion, this protocol analyzes the function of blood bank platelets and this ultimately aids in optimization of the processing chain including phlebotomy, transport, component preparation, storage and transfusion.

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Platelet transfusion and hemostasis was modeled using blood reconstitution and microfluidic flow chambers to investigate the function of blood banking platelets. The data demonstrate the consequences of platelet storage lesion on hemostasis, in vitro.

微流体流钱伯斯再造使用血止血建模和血小板输注<em&gt;体外</em

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of ...

A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is restricted to platelet deposition only. The intricate relationship between Ca2+-dependent coagulation and platelet function requires careful and controlled recalcification of blood prior to analysis. Our setup uses a Y-shaped mixing channel, which supplies concentrated Ca2+/Mg2+ buffer to flowing blood just prior to perfusion, enabling rapid recalcification without sample stasis. A ten-fold difference in flow velocity between both reservoirs minimizes dilution. The recalcified blood is then perfused in a collagen-coated analysis chamber, and differential labeling permits real-time imaging of both platelet and fibrin deposition using fluorescence video microscopy. The system uses only commercially available tools, increasing the chances of standardization. Reconstitution of thrombocytopenic blood with platelets from banked concentrates furthermore models platelet transfusion, proving its use in this research domain. Exemplary data demonstrated that coagulation onset and fibrin deposition were linearly dependent on the platelet concentration, confirming the relationship between primary and secondary hemostasis in our model. In a timeframe of 16 perfusion min, contact activation did not take place, despite recalcification to normal Ca2+ and Mg2+ levels. When coagulation factor XIIa was inhibited by corn trypsin inhibitor, this time frame was even longer, indicating a considerable dynamic range in which the changes in the procoagulant nature of the platelets can be assessed. Co-immobilization of tissue factor with collagen significantly reduced the time to onset of coagulation, but not its rate. The option to study the tissue factor and/or the contact pathway increases the versatility and utility of the assay.

This paper describes an experimental model of hemostasis that simultaneously measures platelet function and coagulation. Platelet and fibrin fluorescence is measured in real-time, and platelet adhesion rate, coagulation rate, and onset of coagulation are determined. The model is used to determine platelet procoagulant properties under flow in concentrates for transfusion.

微流体流室模型血小板输注与止血措施，实时血小板沉积和纤维蛋白形成

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.