Parte 1: Preparação dos canais microfluídicos de uma placa BioFlux. <ol><li> Antes de executar o experimento célula de fluxo, é preciso preparar os canais microfluídicos com uma camada de proteína de interesse. Neste caso, o colágeno I será utilizado. Cada canal tem uma entrada e uma saída bem. Para esta placa, a entrada também é a esquerda bem alimentar o canal ea saída está bem à direita. </li><li> Diluir o colágeno I revestimento (5mg/ml de ações) para a concentração 200μg/ml em ácido acético 0,02. Será necessário 20μl para cada canal utilizado. Misture por triteration gentil com uma ponta de micropipeta. </li><li> Adicionar 20 l de revestimento para cada canal respectivo para ser utilizado. Deve-se dispensar o líquido para dar um soco interior da tomada assim alimentar o canal de microfluídica de interesse usando uma micropipeta. Evitar a introdução de bolhas, não empurre o ar para fora da pipeta. Incluir um canal sem colágeno para um controle de colágeno não (controle negativo para a adesão e agregação). </li><li> Anexar a interface para a placa de primeiro dedo de apertar os 4 parafusos e, em seguida, uma vez que está tudo pronto, use o driver de torque para apertar completamente. O motorista torque vai clicar quando atinge a tensão máxima. </li><li> Usando o modo manual no software BioFlux, aplicar perfusão para os canais de interesse em 2dyn/cm 2-o da tomada também. Aqui é preciso prestar atenção para o soco oposto interior para ser preenchido com líquido. Isso levará alguns minutos. Um pode assistir a este ocorrer a partir da objetiva de baixa potência em um microscópio (4X) e encontrar a entrada de bem ou segurando o prato e olhando para os poços de entrada de baixo. Deve-se primeiro ver uma conta minúscula de líquido que enche lentamente o soco interior. Uma vez que o soco de entrada interior está cheio, pare de perfusão de uma só vez, empurrando parar no software. </li><li> Incubar a placa em temperatura ambiente por uma hora. </li><li> Remover a interface e adicionar 1 ml de PBS (mais Ca 2 + / Mg 2 +) para a saída bem. Iniciar a perfusão da tomada bem na 2dyn/cm 2. Continue perfusão por 10 minutos. Pare de perfusão e remover a interface. </li><li> Retire o excesso de PBS tanto de entrada e saída de poços - nunca remover o líquido que preenche o soco interior. Bloquear os canais utilizando solução de bloqueio (0,5% v / v BSA em PBS (mais Ca 2 + / Mg 2 +)). Adicionar 1 ml de solução de bloqueio para cada saída também para ser usado e perfundir da tomada bem na 2dyn/cm 2. Continue perfusão por 10 minutos. Pare de perfusão e remover a interface. Canais preparados até esta etapa pode ser usado durante todo o dia. </li><li> Antes de adicionar o sangue, remover o líquido em ambos os lados do canal, exceto para os socos interior. </li></ol> Parte 2. Preparando o sangue com GPIIb / IIIa de anticorpos inibidores. <ol><li> Enquanto que o colágeno é incubar nos canais, é preciso preparar o sangue total. Deve-se seguir as normas de biossegurança ditada por seu / sua instituto ao manipular sangue humano. </li><li> Sangue humano fresco (de um indivíduo em jejum), feita em anticoagulante citrato de sódio deve ser usado dentro de 3 horas após a coleta. </li><li> Prepare sangue adicionando calceína 4μM AM. Prepare um estoque 4mM de calceína AM em DMSO e adicionar sangue numa diluição de 1 / 1000 v / v. Misture por inversão suave. </li><li> Dispensar 1 ml da calceína AM rotulados de sangue em 10 - 1,5 ml microtubos. Adicionar GPIIb / IIIa anticorpo inibidor para cada na diluição desejada (o peso molecular de IgG é de ~ 150kDa). Uma série de diluição exemplo é de 1200nm para 9nM e inclui um controle de anticorpos não e um controle de anticorpos não relacionados. Um excelente controle positivo para a inibição seria ReoPro (abciximab, Eli Lilly) em uma concentração de 20ug/ml. Misture por inversão suave. </li><li> Incubar a temperatura ambiente por 1 hora. Misture por inversão suave a cada 10 minutos. </li></ol> Parte 3: A execução do experimento de célula de fluxo na estação de trabalho BF1000. <ol><li> Primeiro, é preciso estabelecer um protocolo automatizado no módulo de controle BioFlux. Para colágeno I, deve-se estabelecer um protocolo para executar 10dyn/cm 2 para 10 minutos da tomada também. Neste ponto, deve-se também configurar os parâmetros de aquisição de dados usando a estação de trabalho BF1000, incluindo posições de canal, captura em comprimento de onda FITC e lapso de tempo da informação. Recomenda-se a captura de três campos de vista usando uma objetiva de 10x por canal a cada 30 segundos sobre uma duração de 10 minutos no total. </li><li> Trabalhar rapidamente lugar, 500ul de sangue preparada de cada condição em separado canais preparados. Lugar nenhum sangue controle de anticorpos em um canal que é revestido com colágeno e um que é apenas bloqueado. </li><li> Lugar da interface na placa. </li><li> Coloque a placa no microscópio workstation BF1000. Grampo na interface. </li><li> Executar o ajuste de carga de placa. Além disso, mover palco para um sangue bem que o contém. Set FTImagem IC parâmetros de captura (tempo de exposição, etc ... ganham). </li><li> No software de montagem BioFlux, iniciar a aquisição de começar o fluxo e aquisição simultaneamente. </li><li> Retirar a placa da estação de trabalho BF1000, dispor de placa de acordo com as diretrizes institucionais. </li></ol> Parte 4: Resultados Representante. Aqui, o protocolo de adesão e agregação plaquetária em canais microfluídicos foi apresentado. Tratamento com um inibidor da agregação plaquetária, anti-GPIIb/IIIa também foi incluído no protocolo. Usando colágeno revestido canais microfluídicos do sistema BioFlux, deve-se esperar para ver a formação de trombos agressivo ao longo do tempo com a amostra não tratada de controle de sangue e não a formação de trombos no canal sem revestimento. Em um experimento realizado recentemente, o tamanho médio de agregados em condições de controle foi 2000μm 2. Figura 1. <img src="/files/ftp_upload/1644/1644fig1.jpg" alt="Figura 1" /> Ativado GPIIb / IIIa é potente mediador de plaquetas plaquetas interações e estabilização de agregação 1. Adesão ao colágeno ativa o complexo GPIIb / IIIa para obter esta resposta 2. Após a incubação com anti-GPIIb/IIIa por 1 hora antes da exposição ao cisalhamento, deve-se esperar para observar uma diminuição no tamanho dos trombos, assim como uma diminuição na freqüência de formação de trombos. Uma resposta dose-dependente geralmente pode ser observado em 10dyn/cm 2. Figura 2. <img src="/files/ftp_upload/1644/1644fig2.jpg" alt="Figura 2" /> O IC 50 valor para este inibidor específico foi 17nM a 10 dyn / cm 2. Inibição máxima (para o doador) em relação ao controle de nenhum anticorpo foi de 11% a 10 dyn / cm 2. Embora os métodos aqui foram apresentados usando uma proteína da matriz extracelular específica e um inibidor específico da agregação plaquetária, o protocolo é extensível a outros revestimentos, outros tipos de células e outros inibidores para ensaios de adesão celular. Os ingredientes chave para o sucesso de tais experiências são evitar bolhas nos canais microfluídicos, diluição correta de todos os reagentes na diluentes correta, e condições de incubação adequados para todos os reagentes.

<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>

Os autores que contribuem são funcionários da Fluxion Biosciences.

Embora os métodos aqui foram apresentados usando uma proteína da matriz extracelular específica e um inibidor específico da agregação plaquetária, o protocolo é extensível a outros revestimentos, outros tipos de células e outros inibidores para ensaios de adesão celular. Os ingredientes chave para o sucesso de tais experiências são evitar bolhas nos canais microfluídicos, diluição correta de todos os reagentes na diluentes correta, e condições de incubação adequados para todos os reagentes.

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		<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

Agregação plaquetária ocorre em resposta à lesão vascular, onde a matriz extracelular abaixo do endotélio tem sido exposto. A cascata de adesão de plaquetas ocorre na presença de fluxo de cisalhamento, não um fator contabilizado em convencional (estática) bem-placa ensaios. Este artigo relata um ensaio de agregação plaquetária, utilizando um formato de prato bem-microfluídicos para emular condições fisiológicas do fluxo de cisalhamento. Proteínas extracelular, colágeno I ou fator de von Willebrand são depositados dentro do canal microfluídicos com uso de perfusão ativa, com uma bomba pneumática. As proteínas da matriz são, então, lavados com buffer e bloqueados para preparar o canal microfluídicos para interações de plaquetas. Sangue total marcada com corante fluorescente é perfundido através do canal em várias taxas de fluxo, a fim de conseguir a ativação e agregação plaquetária. Inibidores da agregação plaquetária pode ser adicionada antes do experimento célula de fluxo para gerar IC50 dados de resposta de dose.

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

A cascata de adesão de plaquetas ocorre na presença de fluxo de cisalhamento, não um fator contabilizado em convencional (estática) bem-placa ensaios. Este artigo relata um ensaio de agregação plaquetária, utilizando um formato de prato bem-microfluídicos para emular condições fisiológicas do fluxo de cisalhamento.

Adesão e agregação plaquetária sob fluxo usando células de fluxo microfluídicos

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 visualizações.  Fluxion Biosciences, Inc.. A cascata de adesão de plaquetas ocorre na presença de fluxo de cisalhamento, não um fator contabilizado em convencional (estática) bem-placa ensaios. Este artigo relata um ensaio de agregação plaquetária, utilizando um formato de prato bem-microfluídicos para emular condições fisiológicas do fluxo de cisalhamento.

Vídeo: 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.

Purificação de Citometria de Fluxo de células de camundongo meiótica

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 ...

Biologia

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.

Optimizado coloração e Proliferação Métodos de Modelagem para Monitoramento Divisão Celular usando Corantes Rastreamento celulares

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

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.

Medindo intracelular de Ca<sup&gt; 2 +</sup&gt; Mudanças no esperma humano, utilizando quatro técnicas: fluorometria convencional, Parado Fluorometria fluxo, citometria de fluxo e única célula de imagem

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.

De gravidade reduzida Manifestações ambiente de hardware de uma tecnologia de mistura de fluxo Cytometer e Companion microfluídicos Prototype Miniaturized

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.

Medidas cálcio citosólico em células epiteliais renais por citometria de fluxo

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 Thermo Flow - Combinando optogenética com Citometria de Fluxo

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.

Análise de suspensões celulares isoladas de tecidos sólidos por citometria de fluxo espectral

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.

Imagens de células vivas de degradação e secreção de plaquetas sob fluxo

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.

Quantificação de Densidades Celulares e Propriedades Antigênicas usando Levitação Magnética

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

Immunophenotyping and Cell Sorting of Human MKs from Human Primary Sources or Differentiated In Vitro from Hematopoietic Progenitors

Megakaryocyte (MK) differentiation encompasses a number of endomitotic cycles that result in a highly polyploid (reaching even&#160;&#62;64N) and extremely large cell (40-60 &#181;m). As opposed to the fast-increasing knowledge in megakaryopoiesis at the cell biology and molecular level, the characterization of megakaryopoiesis by flow cytometry is limited to the identification of mature MKs using lineage-specific surface markers, while earlier MK differentiation stages remain unexplored. Here, we present an immunophenotyping strategy that allows the identification of successive MK differentiation stages, with increasing ploidy status, in human primary sources or in vitro cultures with a panel integrating MK specific and non-specific surface markers. Despite its size and fragility, MKs can be immunophenotyped using the above-mentioned panel and enriched by fluorescence-activated cell sorting under specific conditions of pressure and nozzle diameter. This approach facilitates multi-Omics studies, with the aim to better understand the complexity of megakaryopoiesis and platelet production in humans. A better characterization of megakaryopoiesis may pose fundamental in the diagnosis or prognosis of lineage-related pathologies and malignancy.

Immunophenotyping and Cell Sorting of Human MKs from Human Primary Sources or Differentiated In Vitro from Hematopoietic Progenitors

Here, we present an immunophenotyping strategy for the characterization of megakaryocyte differentiation, and show how that strategy allows the sorting of megakaryocytes at different stages with a fluorescence-activated cell sorter. The methodology can be applied to human primary tissues, but also to megakaryocytes generated in culture in vitro.