This work was supported in part by the National Science Foundation (CBET 0846453).

1. Fabrication of silicon masters using standard SU-8 photolithography1(not shown in this video).
 <ol>
<li>Use standard SU-8 photolithography methods to create a SU-8 &quot;master&quot; (SU-8 2025, MicroChem, Newton, MA) for fabricating the PDMS mold that contains the microfluidic network and chemotaxis chamber. Such master molds can be fabricated at any microfabrication facility (e.g., Stanford Microfluidics Foundry; <a href="http://thebigone.stanford.edu/foundry/">http://thebigone.stanford.edu/foundry/</a>). </li>
<li>Prior to PDMS replication, expose the SU-8 master to fluorosilane vapor in a desiccator attached to a vacuum source to facilitate easy release of PDMS from the master. Add a drop of fluorosilane to a paper towel and apply vacuum for one min. Remove the vacuum and allow 30 min for deposition of fluorosilane. Keep the SU-8 master in a closed container for future use. </li>
 </ol>
2. Replica molding of PDMS from SU-8 master: The fluidic layer and the control layer are made by replica molding of PDMS from SU-8 master.
<ol>
<li>Mix the PDMS pre-polymer and cross-linker at a 10:1 wt. ratio and degas in a dessicator for 1 hour (or until air bubbles are removed from the PDMS mixture).</li>
<li>Place the SU-8 master in a petri dish and carefully pour the PDMS mixture on top of the SU-8 master to the desired thickness.</li>
<li>Heat the petri dish containing the PDMS and the SU-8 master mold at 80&deg;C for two hours to cure the PDMS. </li>
<li>Remove the cured PDMS-covered SU-8 master from the hotplate and peel the PDMS mold from the SU-8 master. The desired structure will be embedded in the PDMS mold.</li>
<li>Punch holes in the PDMS mold for tubing to the gradient generator and cell inlet using a 20 gauge blunt-end needle.</li>
 </ol>
3. Bonding of PDMS devices:
 <ol>
<li>Clean a glass microscope slide with isopropanol and dry with nitrogen or an air stream.</li>
<li>Expose the PDMS and glass slide to oxygen plasma in a plasma asher. </li>
<li>Bring the PDMS mold into contact with the glass slide. Heat the contacted slide and PDMS mold to 65&deg;C for 15 min. </li>
</ol>
4. Assembling the PDMS device
<ol>
 <li>Using a razor blade, cut tubing at a 45&deg; angle to the correct length required for the device.</li>
 <li>Insert one end of the tubing into the holes punched in the device (e.g., outlet) using forceps.</li>
 <li>Using forceps, insert a blunt 30-gauge needle into the other end of the tubing.</li>
 <li>Repeat step 4.2 for all the remaining holes.</li>
 <li>Collect 1mL of chemotaxis buffer (CB) in a 3mL syringe, taking care to remove air bubbles from the syringe.</li>
 <li>Fix a needle hub to one end of the outlet tubing.</li>
 <li>Add CB to the needle hub, and tap the needle hub to remove any air bubbles.</li>
 <li>Push a little CB out of the tip of the syringe and connect the 3mL syringe to the needle hub without trapping air.</li>
 <li>Push CB through the device until all remaining needle hubs are filled with with CB. This should remove a majority of the air bubbles.</li>
 <li>Fill two 500&#956;L syringes with CB containing the appropriate concentrations of the chemoeffector being tested. Eliminate air bubble formation by pushing out a small drop of the syringe contents and touching the drop to the liquid in the needle hub and attaching. </li>
 <li>Similarly, connect a syringe containing CB to the bacteria inlet. This will be removed when bacteria are introduced into the device. </li>
</ol>
5. Growth of highly motile E. coli
<ol>
<li>Grow overnight cultures of E. coli RP437 with a GFP expression plasmid in 20 mL of tryptone broth (TB) at 32&deg;C with shaking. Add appropriate concentrations of antibiotic to maintain the plasmid. In our protocol, we use a low-copy plasmid pCM182 for detecting bacteria based on fluorescence in the device. For E. coli, growing cultures at 32&deg;C is recommended for high motility, as motility is lower at higher temperatures. </li>
<li>Use the overnight culture to inoculate a 20 mL culture of TB in a 250 mL Erlenmeyer flask to an optical density at 600nm of ~ 0.05. Grow the culture with shaking at 32&deg;C. </li>
<li>Harvest the cells at an OD600 of ~0.35 0.45 by low-speed centrifugation at 400 x g for 5 min. </li>
<li>Resuspend cells to an OD600 of ~0.35 in CB containing the chemoeffector at the concentration expected in the middle of the channel. </li>
<li>Add RFP-labeled dead cells at an OD600 of ~ 0.35 to the GFP-expressing live cells. The dead (red) cells are used as an internal control to ensure that any migration is not due to flow effects.</li>
</ol>
6. Monitoring chemotaxis
<ol>
<li>Remove some of the CB from the cell inlet needle hub. Refill with the cell suspension.</li>
<li>Gently fill the 50&#956;L syringe with the resuspended cells. This step needs to be done slowly as flagella can be sheared and will reduce motility.</li>
<li>Attach the 50&#956;L syringe to the inlet-needle hub as described in steps step 4.10.</li>
<li>Position the device on the stage of a fluorescence microscope equipped with a high-speed camera for image acquisition. Place the inlet syringes (both the 500 &mu;L gradient syringes and the 50 &mu;L cell syringe) into the syringe pump and initiate flow such that the gradient is formed and cells are flowing through the tubing. </li>
<li>Once cells enter the chemotaxis chamber, wait ~ 20 min for the system to stabilize before imaging. </li>
<li>Collect green (live) and red (dead) fluorescence images at different locations along the length of the chamber (corresponding to different exposure times). Typically, we collect 100 images at each location at 3 sec intervals. </li>
</ol>
7. Data analysis: The following steps can be performed using any commercially available image analysis program (e.g., ImageJ, Metamorph) or using simple codes written in Matlab.
<ol>
<li>Remove the background pixels in the image (i.e., set threshold pixel intensity and remove all pixels that have intensity less than the threshold). This minimizes noise in the analysis.</li>
<li>Use the position of the dead cells (red fluorescence) to determine the center of the image. Since the dead cells show no chemotaxis, this represents the position where cells entered the chemotaxis chamber and would be detected in the absence of any migration.</li>
<li>Locate live cells (green fluorescence) in the image. </li>
<li>Divide the image into channels and determine the number of live cells in each channel. In our prior work3, the 1050 &mu;m wide chemotaxis chamber was divided into 64 channels that are each ~ 16 &mu;m wide. </li>
<li>Repeat steps 7.1 7.3 for all images and sum the total cell counts for each channel across all images. This gives the total cell count detected at each channel over the duration of the experiment.</li>
<li>Calculate the chemotaxis partition coefficient (CPC), which represents the direction of migration, as follows: Each cell located in the high concentration (right) and low concentration (left) sides of the dead cells is assigned a multiplier of +1 and -1, respectively. That is, a cell that migrates up a gradient is multiplied by +1 while cells moving down the gradient are multiplied by -1. All the multiplied values are added up and normalized to the total number of detected cells. For example, a CPC value of 0.40 indicates that 40% more of the total bacteria moved to the high concentration side than the low concentration side (i.e., the chemoeffector is an attractant as more bacteria were detected moving up the gradient). </li>
<li>Calculate the chemotaxis migration coefficient (CMC), which weighs the migration of cells by the distance travelled, as follows: Weight the cell count in each channel by a factor that is proportional to the distance that cells migrate. A cell that moves to the farthest high-concentration position (channel 64) is given a weighting factor of +1, whereas one that moves halfway into the higher concentration side is given a weighting factor of +0.5, and a cell moving to the farthest low-concentration position is weighted by -1. Sum up all the weighted cell counts and normalize to the number of cells to generate the CMC. </li>
</ol>
Representative Results3,4:
We have used the microfluidic device (Figure 1A) described here to investigate chemotaxis of E. coli in gradients of attractants (aspartic acid, autoinducer-2) and repellents (NiSO4, indole)3,4. The prototypical chemotaxis strain5 E. coli RP437 expressing GFP was used, along with kanamycin-killed E. coli TG1 cells expressing red fluorescent protein to monitor flow effects. The concentration gradient formed in the &mu;Flow device is shown in Figure 1B. The cell inlet was capped so that there was no flow through it and a stable gradient of 0 100 ng/mL of fluoresscein was established in the device. The pixel intensity across the device was used to determine the concentration profile of fluorescein. The data show that bacteria encounter a linear gradient when they enter the chemotaxis chamber. Figure 2A and B shows fluorescence images of E. coli RP437 migrating in response to gradients of aspartic acid (0 100 &mu;M) and NiSO4 (0 225 &mu;M). The observed responses to these canonical attractant and repellent are as predicted. Figure 3A shows the quantified distribution of E. coli RP437 in the absence of a concentration gradient (i.e., null gradient of aspartic acid), while Figures 3B and C show the distribution in gradients of aspartic acid and NiSO4. The specificity of these responses (i.e., the bias in migration) is evident as a E. coli RP437 &Delta;tar strain (i.e., strain lacking the Tar chemoreceptor that is used in E. coli to sense aspartic acid and nickel) does not demonstrate the bias in migration in the presence of a concentration gradient of aspartic acid or NiSO4. The trends evident in the spatial distribution profiles are also consistent with the calculated CPC and CMC values. The CPC values for the migration of E. coli RP437 in gradients of aspartic acid or NiSO4 are +0.33 and -0.33, respectively. The corresponding CMC values are +0.13 and -0.14, respectively. In contrast, the CPC and CMC values with the E. coli RP437 &Delta;tar strain in gradients of aspartic acid or NiSO4 are negligible. In addition, the CPC and CMC in a null gradient of aspartic acid are +0.03 and +0.02, respectively, and indicate the lack of bias in migration in the absence of a gradient.
<img src="/files/ftp_upload/1779/1779fig1a.jpg" alt="Figure 1a" /> <img src="/files/ftp_upload/1779/1779fig1b.jpg" alt="Figure 1b" /> Figure 1. Device schematic and concentration gradient. 
(A) Schematic representation of the microfluidic chemotaxis device. The device consists of a gradient-mixing module (20 x 100 x 18750 &mu;m) and a chemotaxis observation module (20 x 1050 x 11500 &mu;m). The widths of the two gradient channels entering the device are 500 &mu;m and the width of the bacteria inlet is 50 &mu;m. The inset depicts a gradient of signaling molecule (grey) and bacteria migrating in response to it. Live bacteria are depicted as solid ovals, whereas dead bacteria are shown as open ovals. (B) A concentration gradient between 0 and 100 ng/mL fluorescein was generated in the device and imaged using fluorescence microscopy. Fluorescence images were acquired after 30 min and quantified by image analysis.
<img src="/files/ftp_upload/1779/1779fig2.jpg" alt="Figure 2" /> Figure 2. Chemotaxis of E. coli RP437 in the microfluidic device. 
E. coli RP437 was exposed to a gradient of (A) 0-100 &mu;M L-aspartate or (B) 0-225 &mu;M NiSO4 in the device and the migration of live bacteria (green) towards L-aspartate or away from nickel imaged every 2.5 sec for 30 min. Dead bacteria (red) served as the control for flow effects in the device. Images were quantified using an in-house developed analysis program3. Data shown are representative pseudo-colored images from three independent experiments.
<img src="/files/ftp_upload/1779/1779fig3a.jpg" alt="Figure 3a" /> <img src="/files/ftp_upload/1779/1779fig3b.jpg" alt="Figure 3b" /> <img src="/files/ftp_upload/1779/1779fig3c.jpg" alt="Figure 3c" /> Figure 3: Quantification of migration profiles to a canonical attractant and repellent. 
Spatial distribution of RP437 in the microfluidic device in response to (A) uniform 100 &mu;M L-aspartate (i.e., no gradient), (B) a 0 100 &mu;M gradient of aspartate, and (C) a 0 225 &mu;M gradient of NiSO4. The distribution of dead cells is shown as a solid line, the distribution of RP437 cells is shown as a dashed line, and the distribution of RP437 eda+ &Delta;tar cells is shown as a dotted line. Data shown are averaged for three independent experiments.

<ol>
	<li>McDonald, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prototyping+of+microfluidic+devices+in+poly(dimethylsiloxane)+using+solid-object+printing.">Prototyping of microfluidic devices in poly(dimethylsiloxane) using solid-object printing.</a> Anal Chem. 74, 1537-1545 (2002).</li><li>Hansen, M. C., Palmer, R. J. J., Udsen, C., White, D. C., Molin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+GFP+fluorescence+in+cells+of+Streptococcus+gordonii+under+conditions+of+low+pH+and+low+oxygen+concentration.">Assessment of GFP fluorescence in cells of Streptococcus gordonii under conditions of low pH and low oxygen concentration.</a> Microbiol. 147, 1383-1391 (2001).</li><li>Englert, D. L., Manson, M. D., Jayaraman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Flow-Based+Microfluidic+Device+for+Quantifying+Bacterial+Chemotaxis+in+Stable,+Competing+Gradients.">A Flow-Based Microfluidic Device for Quantifying Bacterial Chemotaxis in Stable, Competing Gradients.</a> Appl Environ Microbiol. 75, 4557-4564 (2009).</li><li>Englert, D. L., Jayaraman, A., Manson, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Microfluidic techniques for the analysis of bacterial chemotaxis. , (2009).</li><li>Mao, H., Cremer, P. S., &amp;amp, M. a. n. s. o. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive,+versatile+microfluidic+assay+for+bacterial+chemotaxis.">A sensitive, versatile microfluidic assay for bacterial chemotaxis.</a> PNAS. 100 (9), 5449-5454 (2003).</li></ol>

The chemotaxis partition coefficient (CPC) and the chemotaxis migration coefficient (CMC) can be calculated as described in Mao et al5. If a cell is detected on the high concentration side, it is given a value of +1, whereas a cell detected on the low concentration side is given a value of -1. The values are summed up and divided by the total number of cells to generate the CPC. The sign of the CPC (positive or negative) gives the direction of migration (towards or away from a signal). Although the C...

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.

Watch this Scientific Journal Video about A Microfluidic Device for Quantifying Bacterial Chemotaxis in Stable Concentration Gradients at JoVE.com

A Microfluidic Device for Quantifying Bacterial Chemotaxis in Stable Concentration Gradients

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

Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Bacteria constantly monitor the ...

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12.1K visualizações.  Texas A&M University. Este protocolo descreve o desenvolvimento de um dispositivo micro para investigar quimiotaxia bacteriana em gradientes de concentração estável de chemoeffectors.

Vídeo: A Microfluidic Device for Quantifying Bacterial Chemotaxis in Stable Concentration Gradients

Studies of Bacterial Chemotaxis Using Microfluidics - Interview

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.

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.

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.

 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.

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

CD4+ T-Lymphocyte Capture Using a Disposable Microfluidic Chip for HIV

Cell Capture Using a Microfluidic Device

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.

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

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

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

A microfluidic islet perifusion device was developed for the assessment of dynamic insulin secretion of multiple islets and simultaneous fluorescence imaging of calcium influx and mitochondrial potential changes. The device consists of three layers: first layer contains an array of microscale wells (500 &mu;m diameter and 150 &mu;m depth) that help to immobilize the islets while exposed to flow and maximize the exposed surface area of the islets; the second layer contains a circular perifusion chamber (3 mm deep, 7 mm diameter); and the third layer contains an inlet-mixing channel that fans out before injection into the perifusion chamber (2 mm in width, 19 mm in length, and 500 &mu;m in height) for optimizing the mixing efficiency prior to entering the perifusion chamber. The creation of various glucose gradients including a linear, bell shape, and square shapes also can be created in the microfluidic perifusion network and is demonstrated.

A microfluidic islet perifusion device was developed for the assessment of dynamic insulin secretion of multiple islets and simultaneous fluorescence imaging of calcium influx and mitochondrial potential changes.

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