第1部分：一个BioFlux板块的微流体通道的准备工作。 <ol><li>运行流细胞实验之前，需要准备与有兴趣的蛋白质涂层的微流体通道。在这种情况下，胶原蛋白，我将使用。每个通道都有一个入口和一个电源插座。对于这个板块，入口左侧，以及喂养的通道和出口的权利。 </li><li> 200μg/ml浓度0.02M醋酸稀释的Ⅰ型胶原（5mg/ml股票）涂层。一是将需要为每个使用的通道20μL。用微吸管尖混合温柔triteration。 </li><li>添加20μL涂布到每个要使用相应的通道。必须免除液体以及喂养的微流体通道，使用微量的利益插座内打孔。避免引入气泡，不推空气的吸管。包括一个无胶原控制通道无胶原蛋白（阴性对照的粘附和聚集）。 </li><li>第一紧缩的4个螺丝，然后一旦所有设置，使用的扭矩驱动程序完全收紧的手指，将接口板。扭矩驱动程序，单击时达到最大的密封性。 </li><li>在BioFlux软件中使用手动模式，适用于灌注在2dyn/cm 2从电源插座以及利益的渠道。在这里，人们必须留意对面的内心充满液体的一拳。这将需要几分钟。一个可以观看此发生显微镜使用了低功耗的目标（4X），或板和进气口井从下面找到入口。首先应该看到的液体小珠，慢慢填补了内心的一拳。入口内打孔一旦被填满后，立即停止推动软件停止灌注。 </li><li>板在室温下孵育一小时。 </li><li>删除接口和电源插座以及添加1毫升的PBS（加的Ca 2 + / Mg 2 +的 ）。 2dyn/cm 2插座以及从开始灌注。继续灌注10分钟。停止灌注，并删除接口。 </li><li>删除多余的PBS从入口和出口井 - 千万不要去除液体，填补了内心的一拳。用封闭液（0.5％V / V BSA在PBS（加的Ca 2 + / Mg 2 +的 ））座的渠道。加入封闭液1毫升，以每个插座以及用于灌注2dyn/cm 2插座。继续灌注10分钟。停止灌注，并删除接口。编制到这一步的通道，可使用一整天。 </li><li>增加血液之前，取消对通道两侧的液体，除了内在拳。 </li></ol>第2部分。准备与GPIIb / IIIa受体抑制抗体的血。 <ol><li>虽然胶原蛋白是在孵化的渠道，就必须准备的全血。人们应遵​​循由他/她的研究所的生物安全法规处理时口授的人体血液。 </li><li>绘制成柠檬酸钠抗凝新鲜人血（空腹个人）应收集3小时内使用。 </li><li>准备加入4μm的钙黄绿素AM的血。准备一个4mm的股票和钙黄绿素在DMSO上午添加到血液稀释1 / 1000 V / V。混合轻轻翻转。 </li><li>分配1毫升的钙黄绿素AM标示为10血 - 1.5ml的微管。 GPIIb / IIIa抑制剂抗体添加到每个所需的稀释液（IgG的分子量是〜150kDa）。一个例子稀释系列是从1200nm的9nM，包括一个没有抗体控制和无关抗体控制。 一个很好的抑制阳性对照，将ReoPro在20ug/ml浓度（阿昔单抗，礼来公司）。轻轻颠倒混合。 </li><li>室温孵育1小时。轻轻颠倒混合，每10分钟。 </li></ol>第3部分：BF1000工作站上运行流细胞实验。 <ol><li>首先，必须建立在BioFlux控制模块自动协议。 Ⅰ型胶原，应该成立一个协议，运行10分钟从电源插座以及10dyn/cm 2 。在这一点上，人们还应该设置使用BF1000工作站的数据采集参数，包括通道位置，捕捉在FITC标记的波长和时间推移信息。它是建议捕捉到的视场，使用持续时间总共10分钟的每通道10倍的目标，每30秒。 </li><li>工作迅速，成单独编制的渠道，500ul每个条件的准备血液。放置到一个通道，与胶原蛋白和一个只是被阻止涂没有抗体控制血压。 </li><li>放在板的接口。 </li><li>放置盘上BF1000工作站显微镜。钳接口上。 </li><li>执行板负荷调整。此外，移动舞台，一个含有血液。设置金融时报IC图像采集参数（曝光时间，增益等... ...）。 </li><li>在BioFlux蒙太奇软件中，开始收购，同时开始流和收购。 </li><li> BF1000工作站移除板，处理板根据机构指引。 </li></ol>第4部分：代表性的结果。 这里的血小板粘附和聚集在微流体通道的协议。血小板聚集抑制剂的治疗，anti-GPIIb/IIIa也包括在该协议。使用胶原蛋白涂BioFlux系统的微流体通道，应该会看到随着时间的推移积极与未经处理的控制血液样本和没有涂层通道上的血栓形成血栓形成。在最近进行的实验，总量控制的条件下的平均大小为 2000μm 2。 图1。 <img src="/files/ftp_upload/1644/1644fig1.jpg" alt="图1" />活化的GPIIb / IIIa受体是血小板血小板的相互作用和聚合稳定 1有力的调解人。粘附于胶原激活GPIIb / IIIa受体复合物，引起这种反应2。 anti-GPIIb/IIIa孵化前1小时剪切曝光后，应观察减少血栓的大小，以及减少了血栓形成的频率。剂量依赖反应通常可以观察10dyn/cm 2。 图2。 <img src="/files/ftp_upload/1644/1644fig2.jpg" alt="图2" /> 该IC 50值这个特定的抑制剂是17nM于2厘米/ DYN 10。最大抑制（此捐助）相比，没有抗体控制在10 DYN /厘米2为11％。 虽然这里的方法提出了使用特定的细胞外基质蛋白和血小板聚集的特异性抑制剂，该协议是扩展到其他涂料，其他类型的细胞和其他细胞粘附实验抑制剂。这种实验成功的关键因素是避免在微流体通道，在正确的稀释剂所有试剂的正确稀释，和适当的孵化条件，所有的试剂气泡。

<ol>
	<li>Jackson, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+growing+complexity+of+platelet+aggregation.">The growing complexity of platelet aggregation.</a> Blood. 109, 5087-5095 (2007).</li><li>Nakamura, T., Kambayashi, J., Okuma, M., Tandon, N. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+the+GP+IIb-IIIa+complex+induced+by+platelet+adhesion+to+collagen+is+mediated+by+both+alpha2beta1+integrin+and+GP.">Activation of the GP IIb-IIIa complex induced by platelet adhesion to collagen is mediated by both alpha2beta1 integrin and GP.</a> VI. J Biol Chem. 274, 11897-11903 (1999).</li></ol>

撰稿人Fluxion Biosciences公司的员工。

虽然这里的方法提出了使用特定的细胞外基质蛋白和血小板聚集的特异性抑制剂，该协议是扩展到其他涂料，其他类型的细胞和其他细胞粘附实验抑制剂。这种实验成功的关键因素是避免在微流体通道，在正确的稀释剂所有试剂的正确稀释，和适当的孵化条件，所有的试剂气泡。

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>collagen I, Bovine</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>A1064401</td>
<td>other sources of collagen I can be used</td>
</tr>
<tr>
<td>PBS plus Ca/Mg</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>14040-117</td>
<td>&nbsp;</td>
<tr>
<td>Bovine serum albumin (BSA)</td>
<td>Reagent</td>
<td>EMD</td>
<td>EM-2930 Bottom of Form</td>
<td>From VWR</td>
</tr>
<tr>
<td>calcein AM</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>C1430</td>
<td>Calcein AM from BD can also be used</td>
</tr>
<tr>
<td>BioFlux 1000 System</td>
<td>Reagent</td>
<td>Fluxion Biosciences</td>
<td>Contact Company</td>
<td>&nbsp;</td>
<tr>
<td>BioFlux Plate</td>
<td>Reagent</td>
<td>Fluxion Biosciences</td>
<td>900013</td>
<td>&nbsp;</td>
<tr>
<td>antiGPIIb/IIIa</td>
<td>Reagent</td>
<td>Abcam</td>
<td>ab33407</td>
<td>an alternative # is ab15021</td>
</tr>
<tr>
<td>ReoPro</td>
<td>Reagent</td>
<td>Eli Lilly</td>
<td>&nbsp;</td>
<td>by Rx only</td>
</tr>		</tbody>
		</table>
		</div>

platelet adhesion and aggregation under flow using microfluidic flow cells

血小板聚集发生在低于内皮细胞外基质已经暴露的血管损伤。血小板粘附级联剪切流的存在，这个因素不占在常规（静态）以及板的检测。本文报道利用一个微井板格式，模拟生理剪切流条件的检测血小板聚集。胞外蛋白，胶原蛋白I或血管性血友病因子使用气动泵的积极灌注微流体通道内沉积。基质蛋白，然后用缓冲液洗涤，并准备血小板相互作用的微流体通道封锁。用荧光染料标记的是全血灌流通过在不同的流量渠道，以达到血小板活化和聚集。血小板聚集抑制剂，可以添加之前生成的IC50剂量反应数据流细胞实验。

Watch this Scientific Journal Video about Platelet Adhesion and Aggregation Under Flow using Microfluidic Flow Cells at JoVE.com

Platelet Adhesion and Aggregation Under Flow using Microfluidic Flow Cells

血小板粘附级联剪切流的存在，这个因素不占在常规（静态）以及板的检测。本文报道利用一个微井板格式，模拟生理剪切流条件的检测血小板聚集。

根据流量的血小板粘附和聚集，使用微流控流细胞

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion ...

platelet-adhesion-aggregation-under-flow-using-microfluidic-flow

Research

JoVE Journal

Biology

18.0K 次观看.  Fluxion Biosciences, Inc.. 血小板粘附级联剪切流的存在，这个因素不占在常规（静态）以及板的检测。本文报道利用一个微井板格式，模拟生理剪切流条件的检测血小板聚集。

视频: Platelet Adhesion and Aggregation Under Flow using Microfluidic Flow Cells

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

流动小鼠减数分裂细胞的流式细胞仪净化

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

科研

生物学

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

优化细胞分裂监测细胞示踪染料的染色和扩散建模方法

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

4D Imaging of Protein Aggregation in Live Cells

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental conditions and an intracellular environment that is tightly packed, sticky, and hazardous to protein stability1. The ability to dynamically balance protein production, folding and degradation demands highly-specialized quality control machinery, whose absolute necessity is observed best when it malfunctions. Diseases such as ALS, Alzheimer's, Parkinson's, and certain forms of Cystic Fibrosis have a direct link to protein folding quality control components2, and therefore future therapeutic development requires a basic understanding of underlying processes. Our experimental challenge is to understand how cells integrate damage signals and mount responses that are tailored to diverse circumstances.
The primary reason why protein misfolding represents an existential threat to the cell is the propensity of incorrectly folded proteins to aggregate, thus causing a global perturbation of the crowded and delicate intracellular folding environment1. The folding health, or "proteostasis," of the cellular proteome is maintained, even under the duress of aging, stress and oxidative damage, by the coordinated action of different mechanistic units in an elaborate quality control system3,4. A specialized machinery of molecular chaperones can bind non-native polypeptides and promote their folding into the native state1, target them for degradation by the ubiquitin-proteasome system5, or direct them to protective aggregation inclusions6-9.
In eukaryotes, the cytosolic aggregation quality control load is partitioned between two compartments8-10: the juxtanuclear quality control compartment (JUNQ) and the insoluble protein deposit (IPOD) (Figure 1 - model). Proteins that are ubiquitinated by the protein folding quality control machinery are delivered to the JUNQ, where they are processed for degradation by the proteasome. Misfolded proteins that are not ubiquitinated are diverted to the IPOD, where they are actively aggregated in a protective compartment.
Up until this point, the methodological paradigm of live-cell fluorescence microscopy has largely been to label proteins and track their locations in the cell at specific time-points and usually in two dimensions. As new technologies have begun to grant experimenters unprecedented access to the submicron scale in living cells, the dynamic architecture of the cytosol has come into view as a challenging new frontier for experimental characterization. We present a method for rapidly monitoring the 3D spatial distributions of multiple fluorescently labeled proteins in the yeast cytosol over time. 3D timelapse (4D imaging) is not merely a technical challenge; rather, it also facilitates a dramatic shift in the conceptual framework used to analyze cellular structure.
We utilize a cytosolic folding sensor protein in live yeast to visualize distinct fates for misfolded proteins in cellular aggregation quality control, using rapid 4D fluorescent imaging. The temperature sensitive mutant of the Ubc9 protein10-12 (Ubc9ts) is extremely effective both as a sensor of cellular proteostasis, and a physiological model for tracking aggregation quality control. As with most ts proteins, Ubc9ts is fully folded and functional at permissive temperatures due to active cellular chaperones. Above 30 &deg;C, or when the cell faces misfolding stress, Ubc9ts misfolds and follows the fate of a native globular protein that has been misfolded due to mutation, heat denaturation, or oxidative damage. By fusing it to GFP or other fluorophores, it can be tracked in 3D as it forms Stress Foci, or is directed to JUNQ or IPOD.

Cellular viability depends on timely and efficient management of protein misfolding. Here we describe a method for visualizing the different potential fates of a misfolded protein: refolding, degradation, or sequestration in inclusions. We demonstrate the use of a folding sensor, Ubc9ts, for monitoring proteostasis and aggregation quality control in live cells using 4D microscopy.

在活细胞中蛋白质聚集的4D成像

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental ...

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Spermatozoa are male reproductive cells especially designed to reach, recognize and fuse with the egg. To perform these tasks, sperm cells must be prepared to face a constantly changing environment and to overcome several physical barriers. Being in essence transcriptionally and translationally silent, these motile cells rely profoundly on diverse signaling mechanisms to orient themselves and swim in a directed fashion, and to contend with challenging environmental conditions during their journey to find the egg. In particular, Ca2+-mediated signaling is pivotal for several sperm functions: activation of motility, capacitation (a complex process that prepares sperm for the acrosome reaction) and the acrosome reaction (an exocytotic event that allows sperm-egg fusion). The use of fluorescent dyes to track intracellular fluctuations of this ion is of remarkable importance due to their ease of application, sensitivity, and versatility of detection. Using one single dye-loading protocol we utilize four different fluorometric techniques to monitor sperm Ca2+ dynamics. Each technique provides distinct information that enables spatial and/or temporal resolution, generating data both at single cell and cell population levels.

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Intracellular Ca2+ dynamics are very important in sperm physiology and Ca2+-sensitive fluorescent dyes constitute a versatile tool to study them. Population experiments (fluorometry and stopped flow fluorometry) and single cell experiments (flow cytometry and single cell imaging) are used to track spatio-temporal [Ca2+] changes in human sperm cells.

测量细胞内Ca<sup&gt; +</sup在人类精子的变化采用4项技术：传统的荧光，停流荧光，流式细胞术及单细胞成像

Spermatozoa  are male reproductive cells especially designed to reach, recognize and fuse  with the egg. To perform these tasks, sperm cells must be ...

Reduced-gravity Environment Hardware Demonstrations of a Prototype Miniaturized Flow Cytometer and Companion Microfluidic Mixing Technology

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If humans are to travel beyond low Earth orbit, a transition towards space-ready, point-of-care (POC) testing is required. Such testing needs to be comprehensive, easy to perform in a reduced-gravity environment, and unaffected by the stresses of launch and spaceflight. Countless POC devices have been developed to mimic laboratory scale counterparts, but most have narrow applications and few have demonstrable use in an in-flight, reduced-gravity environment. In fact, demonstrations of biomedical diagnostics in reduced gravity are limited altogether, making component choice and certain logistical challenges difficult to approach when seeking to test new technology. To help fill the void, we are presenting a modular method for the construction and operation of a prototype blood diagnostic device and its associated parabolic flight test rig that meet the standards for flight-testing onboard a parabolic flight, reduced-gravity aircraft. The method first focuses on rig assembly for in-flight, reduced-gravity testing of a flow cytometer and a companion microfluidic mixing chip. Components are adaptable to other designs and some custom components, such as a microvolume sample loader and the micromixer may be of particular interest. The method then shifts focus to flight preparation, by offering guidelines and suggestions to prepare for a successful flight test with regard to user training, development of a standard operating procedure (SOP), and other issues. Finally, in-flight experimental procedures specific to our demonstrations are described.

Spaceflight blood diagnostics need innovation. Few demonstrations have been published illustrating in-flight, reduced-gravity health diagnostic technology. Here we present a method for construction and operation of a parabolic flight test rig for a prototype point-of-care flow-cytometry design, with components and preparation strategies adaptable to other setups.

样机小型化流式细胞仪及配套的混合微流控技术降低重力环境硬件演示

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

在肾小管上皮细胞胞浆钙的测量流式细胞仪

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

LED Thermo Flow &#8212; Combining Optogenetics with Flow Cytometry

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the controlled delivery of light to the cell sample and hence the most popular tools for optogenetic studies are microscopy-based cell analyses and in vitro experiments. The flow cytometer has major advantages over a microscope, including the ability to rapidly measure thousands of cells at single cell resolution. However, it is not yet widely used in optogenetics. Here, we present a device that combines the power of optogenetics and flow cytometry: the LED Thermo Flow. This device illuminates cells at specific wavelengths, light intensities and temperatures during flow cytometric measurements. It can be built at low cost and be used with most common flow cytometers. To demonstrate its utility, we characterized the photoswitching kinetics of Dronpa proteins in vivo and in real time. This protocol can be adapted to almost all optically controlled substances and substantially expands the set of possible experiments. More importantly, it will greatly simplify the discovery and development of new optogenetic tools.

LED Thermo Flow &amp;#8212; Combining Optogenetics with Flow Cytometry

Optically controlled substances are powerful tools to study signaling pathways. To expand the spectrum of possible experiments, we developed a device for studying optically controlled substances in real time using flow cytometry: the LED Thermo Flow.

LED热流量 - 结合光遗传学与流式细胞仪

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

通过光谱流式细胞仪由固态组织中分离出悬浮细胞系的分析

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

Live-cell Imaging of Platelet Degranulation and Secretion Under Flow

Blood platelets are essential players in hemostasis, the formation of thrombi to seal vascular breaches. They are also involved in thrombosis, the formation of thrombi that occlude the vasculature and injure organs, with life-threatening consequences. This motivates scientific research on platelet function and the development of methods to track cell-biological processes as they occur under flow conditions.
A variety of flow models are available for the study of platelet adhesion and aggregation, two key phenomena in platelet biology. This work describes a method to study real-time platelet degranulation under flow during activation. The method makes use of a flow chamber coupled to a syringe-pump setup that is placed under a wide-field, inverted, LED-based fluorescence microscope. The setup described here allows for the simultaneous excitation of multiple fluorophores that are delivered by fluorescently labeled antibodies or fluorescent dyes. After live-cell imaging experiments, the cover glasses can be further processed and analyzed using static microscopy (i.e., confocal microscopy or scanning electron microscopy).

This work describes a fluorescence microscopy-based method for the study of platelet adhesion, spreading, and secretion under flow. This versatile platform enables the investigation of platelet function for mechanistic research on thrombosis and hemostasis.

活细胞成像血小板脱水和分泌流量

Blood platelets are essential players in hemostasis, the formation of thrombi to seal vascular breaches. They are also involved in thrombosis, the ...

Quantification of Cellular Densities and Antigenic Properties using Magnetic Levitation

The described method was developed based on the principles of magnetic levitation, which separates cells and particles based on their density and magnetic properties. Density is a cell type identifying property, directly related to its metabolic rate, differentiation, and activation status. Magnetic levitation allows a one-step approach to successfully separate, image and characterize circulating blood cells, and to detect anemia, sickle cell disease, and circulating tumor cells based on density and magnetic properties. This approach is also amenable to detecting soluble antigens present in a solution by using sets of low- and high-density beads coated with capture and detection antibodies, respectively. If the antigen is present in solution, it will bridge the two sets&#160;of beads, generating a new bead-bead complex, which will levitate in between the rows of antibody-coated beads. Increased concentration of the target antigen in solution will generate a larger number of bead-bead complexes when compared to lower concentrations of antigen, thus allowing for quantitative measurements of the target antigen. Magnetic levitation is advantageous to other methods due to its decreased sample preparation time and lack of dependance on classical readout methods. The image generated is easily captured and analyzed using a standard microscope or mobile device, such as a smartphone or a tablet.

This paper describes a magnetic levitation-based method that can specifically detect the presence of antigens, either soluble or membrane-bound, by quantifying changes in the levitation height of capture beads with fixed densities.

使用磁悬浮定量细胞密度和抗原特性

The described method was developed based on the principles of magnetic levitation, which separates cells and particles based on their density and magnetic ...