Este trabalho foi financiado pela AAUW companheirismo internacional para Adeola Adewola, NIH / NCRR (U42RR023245) para Jose Oberholzer, e O Projeto Diabetes Chicago.

A. Microfabricação de 3 camadas dispositivo perifusion microfluídicos Bem fundo Mestre Protocol (150 mM poços profundos) <ol><li> Limpe o wafer usando uma lâmina de barbear, se necessário. Limpe com acetona, metanol, e IPA. Realizar o tratamento de plasma de 50 Watts por 30 s. </li><li> Spin-SU8-100 @ 2000 rpm. [Passo 1: 500 rpm, 10 s, 100 Passo rpm / s, 2: 2000 rpm, 30 s, a 300 rpm / s]. [NOTA: Não segure o wafer com uma pinça, após girar SU8] </li><li> Bake mole do wafer a 65 ° C por 20 min e aos 95 ° C por 50 min. </li><li> O wafer é exposto à luz UV através de uma máscara desejada. Dose para 150 M de altura é de 650 mJ / cm 2. </li><li> Pós-exposição assar o wafer de 65 ° C por 1 min e em 95 ° C por 12 min. </li><li> Desenvolver o wafer em SU8 desenvolvedor por 15 min. </li></ol> Microcanais Mestre Protocol (500 mm poços profundos) <ol><li> Limpe o wafer usando uma lâmina de barbear, se necessário. Limpe com acetona, metanol, e IPA. Realizar o tratamento de plasma de 50 Watts por 30 s. </li><li> Spin-SU8-2150 @ 1000 rpm. [Passo 1: 500 rpm, 10 s, 100 Passo rpm / s, 2: 1000 rpm, 30 s, a 300 rpm / s]. </li><li> Bake mole do wafer a 65 ° C por 15 min e aos 95 ° C durante 2 horas e 30 min. </li><li> O wafer é exposto à luz UV através de uma máscara desejada. Dose de exposição para 650 M de altura é de 685 mJ / cm 2. </li><li> Pós-exposição assar o wafer de 65 ° C por 5 min e em 95 ° C por 35 min. </li><li> Desenvolver o wafer em SU8 desenvolvedor por 20-30 min. </li></ol> PDMS preparação da solução <ol><li> Polidimetilsiloxano solução (PDMS) é preparado através de uma profunda mistura de 10 partes de elastômero de silicone com 1 parte de agente de cura de um kit Sylgard padrão 184. </li><li> As bolhas geradas na solução PDMS durante o processo de mistura são removidas usando um exsicador de vácuo. </li><li> A bolha sem solução PDMS é lentamente dispensada na SU8 mestres e uma placa de Petri vazia para a terceira camada. </li><li> A temperatura da placa quente é definido como 75 ° C ea PDMS é curado a esta temperatura durante 2 horas </li><li> Entradas, saídas e poços de câmbio são socou para fora usando o furador de tamanho apropriado. </li><li> As camadas são unidas em uma lâmina de vidro (mm tamanho 0.1) usando um dispositivo handheld plasma. </li></ol> B. configuração Experimental <ol><li> Fluxo de etanol 70% através do dispositivo de micro-para esterilizar. DI fluxo de água para lavar o etanol. Perfundir 50 ml de BSA 0,5% através do dispositivo para evitar adsorção não específica de insulina para as paredes de microcanais. </li><li> O gradiente de rampa de glicose e outros relacionados com gradientes gerados pelo software LabView que se comunica com a seringa bombas são testadas para garantir que a gradientes são estáveis. </li><li> 25-30 ilhotas ratos foram incubados com 5 Fura-2/AM M (um indicador de cálcio, sondas moleculares, CA) e 2,5 mM Rodamina 123 (Rh123, um indicador de potencial mitocondrial, Sigma, MO) por 30 min a 37 ° C em Krebs-Ringer buffer (KRB) contendo 2 mM de glicose </li><li> As ilhotas ratos são cuidadosamente pipetados dispositivo perifusion microfluídicos através da porta de entrada. </li><li> A rede de microfluídica é, então, configuração, ligando à entrada das bombas de seringa usando tubos Tygon e um conector Y ea saída para coletor de fração. O dispositivo fica em um estágio de aquecimento (37 ° C) no microscópio e do tubo de admissão é aquecida numa placa de aquecimento para manter a temperatura da câmara e uma solução a 37 ° C. </li><li> Imediatamente após a instalação, as ilhotas ratos são perifused com KRB contendo 2 mM de glicose por 10 min e, em seguida uma rampa de glicose (2mM 25mM) por 25 min. </li><li> Lapso de tempo imagens são coletadas e analisadas a cada 15 s por software SimplePCI. O perifusate também é coletado a cada minuto através de um coletor de frações para analisar a secreção de insulina utilizando ELISA kit. </li></ol> Resultados representante Ilhotas de rato foram perifused com um gradiente linear de 2-25 mM de glicose. Conforme mostrado na Figura 1, o influxo de cálcio e secreção de insulina são desencadeados após cerca de 13 minutos de perifusion, correspondendo a 6 mM de glicose. Mudanças no potencial mitocondrial são vistos anteriormente como esperado, em cerca de 11 minutos. Estes dados demonstram a vantagem de usar esta rede microfluídicos para caracterizar ilhota fisiologia. <img src="/files/ftp_upload/1649/1649fig1.jpg" alt="Figura 1" /> Figura 1. Respostas fisiológicas de estimulação das células da ilhota.

<ol>
	<li>Mohammed, J. S., Wang, Y., Harvat, T. A., Oberholzer, J., Eddington, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+device+for+multimodal+characterization+of+pancreatic+islets.">Microfluidic device for multimodal characterization of pancreatic islets.</a> Lab Chip. 9, 97-106 (2009).</li></ol>

Sistemas tradicionais de ilhotas perifusion (macroescala e microescala) tem algumas limitações, incluindo complexo de configuração e design, alta requisitos técnicos, e dificuldade para criar gradientes usuário prescritos química no sistema. O sistema perifusion microfluídicos descritas aqui supera estas limitações com geometria simples de projeto e fabricação. Mais importante, este sistema pode ser integrado com abordagem imagens de fluorescência que fornecem como uma ferramenta única para estudar a fisio...

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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>60ml Syringes</td>
<td>&nbsp;</td>
<td>BD</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fura-2 fluorescence dye</td>
<td>&nbsp;</td>
<td>Molecular Probes Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Rhodamine123 Fluorescence dye</td>
<td>&nbsp;</td>
<td>Molecular Probes Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Glucose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Bovine Serum Albumin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>30" Silicone tubings</td>
<td>&nbsp;</td>
<td>Cole Palmer</td>
<td>&nbsp;</td>
<td>1/16 x 1/8in</td>
</tr>
<tr>
<td>1.5ml Eppendorf tubes</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Y-connectors</td>
<td>&nbsp;</td>
<td>Cole Palmer</td>
<td>&nbsp;</td>
<td>1/16&#x201D; &amp; 4mm</td>
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<td>Syringe connectors</td>
<td>&nbsp;</td>
<td>Cole Palmer</td>
<td>&nbsp;</td>
<td>female luer plug 1/16&#x201D;</td>
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<td>Straight connectors</td>
<td>&nbsp;</td>
<td>Cole Palmer</td>
<td>&nbsp;</td>
<td>1/16&#x201D;</td>
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<td>Elbow connector</td>
<td>&nbsp;</td>
<td>Cole Palmer</td>
<td>&nbsp;</td>
<td>1/16&#x201D;</td>
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<td>Havard syringe pump</td>
<td>&nbsp;</td>
<td>Harvard Apparatus</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Perifusion device</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Hot plate</td>
<td>&nbsp;</td>
<td>PMC</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Thermometer</td>
<td>&nbsp;</td>
<td>Omega Engineering Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Fraction collector</td>
<td>&nbsp;</td>
<td>Gibson</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Pippettor</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Inverted epiflorescence microscope</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>IX71</td>
<td>&nbsp;</td>
<tr>
<td>Charge-coupled device</td>
<td>&nbsp;</td>
<td>QImaging</td>
<td>&nbsp;</td>
<td>Retiga-SRV, Fast 1394</td>
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a multi-parametric islet perifusion system within a microfluidic perifusion device

Um dispositivo perifusion microfluídicos ilhota foi desenvolvido para a avaliação dinâmica da secreção de insulina de ilhotas múltiplo e imagens de fluorescência simultânea de influxo de cálcio e mitocondrial possíveis mudanças. O dispositivo consiste de três camadas: a primeira camada contém uma matriz de microescala poços (500 mm e diâmetro de 150 profundidade mm) que ajudam a imobilizar o ilhotas enquanto expostos a fluxo e maximizar a área de superfície exposta das ilhotas, a segunda camada contém uma circular perifusion câmara (3 mm de profundidade, 7 mm de diâmetro), ea terceira camada contém um canal de entrada de mistura que os fãs antes de injeção na câmara perifusion (2 mm de largura, 19 mm de comprimento e 500 mm de altura) para otimizar a eficiência de mistura antes de entrar na câmara de perifusion. A criação de gradientes de glucose diversos, incluindo uma linear forma de sino, e formas quadradas também podem ser criados na rede perifusion microfluídicos e é demonstrada.

Watch this Scientific Journal Video about A Multi-Parametric Islet Perifusion System within a Microfluidic Perifusion Device at JoVE.com

A Multi-Parametric Islet Perifusion System within a Microfluidic Perifusion Device

Um dispositivo perifusion microfluídicos ilhota foi desenvolvido para a avaliação dinâmica da secreção de insulina de ilhotas múltiplo e imagens de fluorescência simultânea de influxo de cálcio e mitocondrial possíveis mudanças.

A Multi-Parametric Sistema Perifusion Islet dentro de um dispositivo Perifusion microfluídicos

A microfluidic islet perifusion device was developed for the assessment of dynamic insulin secretion of multiple islets and simultaneous fluorescence ...

a-multi-parametric-islet-perifusion-system-within-microfluidic

Research

JoVE Journal

Biology

11.8K visualizações.  University of Illinois, Chicago. Um dispositivo perifusion microfluídicos ilhota foi desenvolvido para a avaliação dinâmica da secreção de insulina de ilhotas múltiplo e imagens de fluorescência simultânea de influxo de cálcio e mitocondrial possíveis mudanças.

Vídeo: A Multi-Parametric Islet Perifusion System within a Microfluidic Perifusion Device

Murine Pancreatic Islet Isolation

Isolamento Islet murino de pâncreas

Biologia

Fabrication of a Microfluidic Device for the Compartmentalization of Neuron Soma and Axons

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for culturing neurons. Previously, a silicon wafer was patterned with the design for the neuron microfluidic device using SU-8 and photolithography to create a master mold, or what we simply refer to as a "master". Next, we pour the silicon polymer PDMS on top of the master which is then cured by heating the PDMS to 80&deg;C for 1 hour. The PDMS forms a negative mold of the device. The PDMS is then carefully cut and lifted away from the master. Holes are punched where the reservoirs will be and the excess PDMS trimmed away from the device. Nitrogen is used to blow away any excess debris from the device. At this point the devices are now ready for use and can either bonded to corning No. 1 cover glass with a plasma sterilizer/cleaner or can be reversibly bound to the cover glass by simply placing the device on top of the cover glass. The reversible bonding of the device to glass is covered in a separate video and requires first that the device be sterilized either with 70% ethanol or by autoclaving. Plasma treating sterilizes the devices so no further treatment is necessary. It is, however, important, when plasma-treating the devices, to add liquid to the devices within 10 minutes of the plasma treatment while the surfaces are still hydrophilic. Waiting longer than 10 minutes to add liquid to the device makes it difficult for the liquid to enter the device. The neuron devices are typically plasma-bound to cover glass and 0.5 mg/ml poly-L-lysine (PLL) in pH 8.5 borate buffer is immediately added to the device. After a minimum of 3 hours incubating with PLL, the devices are washed with dH2O water a minimum of 3 times with at least 15 minutes between each wash. Next, the water is removed and fresh media is added to the device. At this point the device is ready for use. It is important to remember at this point to never remove all the media from the device. Always leave media in the main channel.

In this video we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to farbricate a microfluidic device for culturing neurons.

Fabricação de um dispositivo micro para a compartimentação de Neuron Soma e axônios

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for ...

A Microfluidic Device with Groove Patterns for Studying Cellular Behavior

We describe a microfluidic device with microgrooved patterns for studying cellular behavior. This microfluidic platform consists of a top fluidic channel and a bottom microgrooved substrate. To fabricate the microgrooved channels, a top poly(dimethylsiloxane) (PDMS) mold containing the impression of the microfluidic channels was aligned and bonded to a microgrooved substrate. Using this device, mouse fibroblast cells were immobilized and patterned within microgrooved substrates (25, 50, 75, and 100 &mu;m wide). To study apoptosis in a microfluidic device, media containing hydrogen peroxide, Annexin V, and propidium iodide was perfused into the fluidic channel for 2 hours. We found that cells exposed to the oxidative stress became apoptotic. These apoptotic cells were confirmed by Annexin V that bound to phosphatidylserine at the outer leaflet of the plasma membrane during the apoptosis process. Using this microfluidic device with microgrooved patterns, the apoptosis process was observed in real-time and analyzed by using an inverted microscope containing an incubation chamber (37&deg;C, 5% CO2). Therefore, this microfluidic device incorporated with microgrooved substrates could be useful for studying the cellular behavior and performing high-throughput drug screening.

We describe a protocol for the fabrication of microfluidic devices that can enable cell capture and culture. In this approach patterned microstructures such as grooves within microfluidic channels are used to create low shear stress regions within which cell can dock.

Um dispositivo micro com padrões Groove para estudar o comportamento celular

We describe a microfluidic device with microgrooved patterns for studying cellular behavior.  This microfluidic platform consists of a top fluidic channel ...

A Gradient-generating Microfluidic Device for Cell Biology

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an enabling experimental tool, because it can precisely manipulate fluid flows, enable high-throughput experiments, and generate stable soluble concentration gradients.  Compared to conventional gradient generators, poly(dimethylsiloxane) (PDMS)-based  microfluidic devices can generate stable concentration gradients of growth factors with well-defined profiles.  Here, we developed simple gradient-generating microfluidic devices with three separate inlets.  Three microchannels combined into one microchannel to generate concentration gradients.  The stability and shape of growth factor gradients were confirmed by fluorescein isothyiocyanate (FITC)-dextran with a molecular weight similar to epidermal growth factor (EGF).  Using this microfluidic device, we demonstrated that fibroblasts exposed to concentration gradients of EGF migrated toward higher concentrations.  The directional orientation of cell migration and motility of migrating cells were quantitatively assessed by cell tracking analysis.  Thus, this gradient-generating microfluidic device might be useful for studying and analyzing the behavior of migrating cells.

We describe a protocol for the microfabrication of the gradient-generating microfluidic device that can generate spatial and temporal gradients in well-defined microenvironment. In this approach, the gradient-generating microfluidic device can be used to study directed cell migration, embryogenesis, wound healing, and cancer metastasis.

Um dispositivo de geração de Gradient microfluídicos de Biologia Celular

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an ...

Preparing E18 Cortical Rat Neurons for Compartmentalization in a Microfluidic Device

In this video, we demonstrate the preparation of E18 cortical rat neurons. E18 cortical rat neurons are obtained from E18 fetal rat cortex previously dissected and prepared. The E18 cortex is, upon dissection, immediately dissociated into individual neurons. It is possible to store E18 cortex in Hibernate E buffer containing B27 at 4&deg;C for up to a week before the dissociation is performed. However, there will be a drop in cell viability. Typically we obtain our E18 Cortex fresh. It is transported to the lab in ice cold Calcium free Magnesium free dissection buffer (CMFM). Upon arrival, trypsin is added to the cortex to a final concentration of 0.125%. The cortex is then incubated at 37&deg;C for 8 minutes. DMEM containing 10% FBS is added to the cortex to stop the reaction. The cortex is then centrifuged at 2500 rpm for 2 minutes. The supernatant is removed and 2 ml of Neural Basal Media (NBM) containing 2% B27 (vol/vol) and 0.25% Glutamax (vol/vol) is added to the cortex which is then re-suspended by pipetting up and down. Next, the cortex is triturated with previously fire polished glass pipettes, each with a successive smaller opening. After triturating, the cortex is once again centrifuged at 2500 rpm for 2 minutes. The supernatant is then removed and the cortex pellet re-suspended with 2 ml of NBM containing B27 and Glutamax. The cell suspension is then passed through a 40 um nylon cell strainer. Next the cells are counted. The neurons are now ready for loading into the neuron microfluidic device.

In this video we demonstrate the preparation of E18 Cortical Rat Neurons.

Preparando E18 neurônios de rato para Cortical Compartimentação em um dispositivo micro

In this video, we demonstrate the preparation of E18 cortical rat neurons. E18 cortical rat neurons are obtained from E18 fetal rat cortex previously ...

Cell Capture Using a Microfluidic Device

Captura de célula usando um dispositivo micro

Assessing Neural Stem Cell Motility Using an Agarose Gel-based Microfluidic Device

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is largely due to the difficulty with which one can create a cell-compatible and steady microenvironment using conventional microfabrication techniques and materials. We address this problem via the introduction of a novel microfabrication material, agarose gel, as the base material for the microfluidic device. Agarose gel is highly malleable, and permeable to gas and nutrients necessary for cell survival, and thus an ideal material for cell-based assays. We have shown previously that agarose gel based devices have been successful in studying bacterial and neutrophil cell migration2. In this report, three parallel microfluidic channels are patterned in an agarose gel membrane of about 1mm thickness. Constant flows with media/buffer are maintained in the two side channels using a peristaltic pump. Cells are maintained in the center channel for observation. Since the nutrients and chemicals in the side channels are constantly diffusing from the side to center channel, the chemical environment of the center channel is easily controlled via the flow along the side channels. Using this device, we demonstrate that the movement of neural stem cells can be monitored optically with ease under various chemical conditions, and the experimental results show that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells. Motility of neural stem cells is an important biomarker for assessing cells aggressiveness, thus tumorigenic factor3. Deciphering the mechanism underlying NSC motility will yield insight into both disorders of neural development and into brain cancer stem cell invasion.

We demonstrate that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells(NSCs) using a novel agarose gel based microfluidic device. This technology can be readily adaptable to other mammalian cell systems where cell sources are scarce, such as human neural stem cells, and the turn around time is critical.

Avaliar motilidade celular Neural Stem Usando um dispositivo Agarose Gel baseado microfluídicos

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is ...

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to six independent experiments in parallel for higher-throughput. We developed a new fabrication method to create microfluidic devices having both micro and macro scale structures within the same device through a single soft-lithography process, enabling mass fabrication with good repeatability.
The multi-compartment microfluidic co-culture platform is composed of one soma compartment for neurons and six axon/glia compartments for oligodendrocytes (OLs). The soma compartment and axon/glia compartments are connected by arrays of axon-guiding microchannels that function as physical barriers to confine neuronal soma in the soma compartment, while allowing axons to grow into axon/glia compartments. OLs loaded into axon/glia compartments can interact only with axons but not with neuronal soma or dendrites, enabling localized axon-glia interaction studies. The microchannels also enabled fluidic isolation between compartments, allowing six independent experiments to be conducted on a single device for higher throughput.
Soft-lithography using poly(dimethylsiloxane) (PDMS) is a commonly used technique in biomedical microdevices. Reservoirs on these devices are commonly defined by manual punching. Although simple, poor alignment and time consuming nature of the process makes this process not suitable when large numbers of reservoirs have to be repeatedly created. The newly developed method did not require manual punching of reservoirs, overcoming such limitations. First, seven reservoirs (depth: 3.5 mm) were made on a poly(methyl methacrylate) (PMMA) block using a micro-milling machine. Then, arrays of ridge microstructures, fabricated on a glass substrate, were hot-embossed against the PMMA block to define microchannels that connect the soma and axon/glia compartments. This process resulted in macro-scale reservoirs (3.5 mm) and micro-scale channels (2.5 &mu;m) to coincide within a single PMMA master. A PDMS replica that served as a mold master was obtained using soft-lithography and the final PDMS device was replicated from this master.
Primary neurons from E16-18 rats were loaded to the soma compartment and cultured for two weeks. After one week of cell culture, axons crossed microchannels and formed axonal only network layer inside axon/glia compartments. Axons grew uniformly throughout six axon/glia compartments and OLs from P1-2 rats were added to axon/glia compartments at 14 days in vitro for co-culture.

We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method.

Um compartimento Multi-glia do SNC Neuron-Co-cultura plataforma microfluídica

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to ...

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has been proposed that the microenvironment that the pathogen encounters in the commensal layer is important in determining the extent of colonization. Current culture methods for investigating pathogen colonization are not well suited for investigating this hypothesis as they do not enable co-culture of bacteria and epithelial cells in a manner that mimics the GI tract microenvironment. Here we describe a microfluidic co-culture model that enables independent culture of eukaryotic cells and bacteria, and testing the effect of the commensal microenvironment on pathogen colonization. The co-culture model is demonstrated by developing a commensal Escherichia coli biofilm among HeLa cells, followed by introduction of enterohemorrhagic E. coli (EHEC) into the commensal island, in a sequence that mimics the sequence of events in GI tract infection.

This protocol describes a microfluidic co-culture model for simultaneous and localized culture of epithelial cells and bacteria. This model can be used for investigating the role of different soluble molecular signals on pathogenesis as well as screen the effectiveness of putative probiotic bacterial strains.

Microfluídicos Co-cultura de células epiteliais e bactérias para a Investigação Solúvel sinal mediada por interações

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has ...

A Microfluidic Device for Quantifying Bacterial Chemotaxis in Stable Concentration Gradients

Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Bacteria constantly monitor the concentration of specific chemoeffectors by comparing the current concentration to the concentration detected a few seconds earlier. This comparison determines the net direction of movement. Although multiple, competing gradients often coexist in nature, conventional approaches for investigating bacterial chemotaxis are suboptimal for quantifying migration in response to concentration gradients of attractants and repellents. Here, we describe the development of a microfluidic chemotaxis model for presenting precise and stable concentration gradients of chemoeffectors to bacteria and quantitatively investigating their response to the applied gradient. The device is versatile in that concentration gradients of any desired absolute concentration and gradient strength can be easily generated by diffusive mixing. The device is demonstrated using the response of Escherichia coli RP437 to gradients of amino acids and nickel ions.

This protocol describes the development of a microfluidic device for investigating bacterial chemotaxis in stable concentration gradients of chemoeffectors.