Name: preparing e18 cortical rat neurons for compartmentalization in a microfluidic device
Uploaded: 2007-10-01T00:00:00
Duration: 12 min
Description: Watch this Scientific Journal Video about Preparing E18 Cortical Rat Neurons for Compartmentalization in a Microfluidic Device at JoVE.com

Preparing E18 Fetal Rat Cortical Neurons for Compartmentalization
Before starting out, it is important to warm all necessary media and reagents to 37&deg;C.
It is also important to&nbsp;sterilize everything that is used for preparing the cells (e.g., rubber bulbs, tube racks, media bottles, etc.), and that which&nbsp;is placed&nbsp;in the hood,&nbsp;by wiping down with 70% ethanol.
<ol>
<li>Place two pieces of E18 Fetal Rat Cortex (one brain, previously dissected) in a 15 ml tube containing 1 ml ice-cold Calcium-free Magnesium-free dissection buffer.</li>
<li>Add 1 ml of 0.25% Trypsin-EDTA to the cortex in dissection buffer, bringing the final volume to 2 ml and final trypsin concentration to 0.125%.</li>
<li>Place the 15 ml tube in a 37&deg;C water bath for 8 minutes.</li>
<li>During this time, fire polish 3 glass Pasteur pipets, forming successively smaller openings, in a biosafety cabinet to help maintain sterility.</li>
<li>After the 8 minute incubation of the cortex, add 10 ml of DMEM containing 10% FBS to the cortex to help stop the trypsin reaction.</li>
<li>Centrifuge the 15 ml tube containing the cortex and DMEM/10% FBS at 2500 rpm for 2 minutes.</li>
<li>In the biosafety cabinet, remove the supernatant from the cortex using a glass Pasteur pipet with vacuum suction attached. Be careful not to disturb or dislodge the pellet.</li>
<li>Add 1 ml of NMB to the cortex pellet and gently pipet up and down. It is very important to avoid creating air bubbles while pipeting up and down, as the air bubbles can damage the cells by&nbsp;oxidation.</li>
<li>Use the fire-polished Pasteur pipet with the widest opening to triturate the cortex by attaching a sterile rubber bulb to the end and pipeting up and down 5 times, again being careful not to introduce air bubbles. Continue this process&nbsp;with the other 2 glass pipets, each with a decreased opening size.</li>
<li>After trituration, centrifuge down&nbsp;the cells again at 2500 rpm for 2 minutes.</li>
<li>After centrifugation, once again remove the supernatant and resuspend the cell pellet in 2 ml of NBM.</li>
<li>Filter the resuspended cell solution through a 45 um cell strainer.</li>
<li>Stain the cells with Trypan Blue, counted, and loaded into the devices. We typically load 20 ul of cells per device.</li>
</ol>
Note: the final concentration of cells is typically between 2.5 million cells/ml and 8 million cells/ml
Loading the cells
After the cells have been counted, it is time to load the cells in the device:
<ol>
<li>Bring the previously prepared devices containing NBM into the biosafety cabinet to maintain sterility. </li>
<li>Remove excess media from the wells by vacuum suction. However, be careful to avoid removing all of the media --&nbsp;there must be some media in the main channel. </li>
<li>Apply 20 ul of cells to the top left hand reservoir of the device (see diagram if necessary). The cells flow into the device and attach to the PLL treated surface. </li>
<li>After loading, place the devices containing cells back in an incubator for 10 minutes to allow the cells to attach. </li>
<li>After 10 minutes, fill the reservoirs with media and place devices back in the incubator. </li>
</ol>

<ol>
	<li>Park, J. W., Vahidi, B., Taylor, A. M., Rhee, S. W., Jeon, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+culture+platform+for+neuroscience+research.">Microfluidic culture platform for neuroscience research.</a> Nat Protoc. 1, 2128-2136 (2006).</li><li>Taylor, A. M., Blurton-Jones, M., Rhee, S. W., Cribbs, D. H., Cotman, C. W., Jeon, N. L. <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+culture+platform+for+CNS+axonal+injury,+regeneration+and+transport.">A microfluidic culture platform for CNS axonal injury, regeneration and transport.</a> Nat Methods. 2, 599-605 (2005).</li></ol>

Cell densities can be varied depending on the application. However, seeding primary neurons at too low of a density typically leads to cell death. Depending on the incubator and the level of humidity, media may need to be changed in the devices every 2 to 3 days. When changing media, it is important after removing the media from the reservoirs, to never remove media from the main channels. Further, it is good practice to place fresh media in the top wells and allow some fresh media to flow through the device and into the main channels before filling up all the reservoirs.

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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>NBM</td>
<td>medium</td>
<td>Invitrogen</td>
<td>21103-049</td>
<td>Neural Basal Media is obtained by Invitrogen under their Gibco line of cell culture reagents.</td>
</tr>
<tr>
<td>E18 rat embryos</td>
<td>Animal</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Obtained from pregnant Sprague Dawley Rats. The pregnant rat is sacrificed and the E18 fetal rat pups dissected to obtain the E18 fetal rat cortex.</td>
</tr>
<tr>
<td>CMFM dissection buffer HBSS</td>
<td>buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>ice-cold Calcium-free Magnesium-free dissection buffer HBSS based.</td>
</tr>
<tr>
<td>15 ml Tube</td>
<td>Tool</td>
<td>BD Falcon</td>
<td>Falcon No. 352097</td>
<td>Available through Fisher Scientific catalog number 14-959-70C</td>
</tr>
<tr>
<td>0.25% Trypsin-EDTA</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>37&#x02DA;C water bath</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Pasteur pipets</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>&nbsp;</td>
<td>glass</td>
</tr>
<tr>
<td>DMEM</td>
<td>medium</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>serum, used at 10% in DMEM</td>
</tr>
<tr>
<td>centrifuge</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>set at 2500 rpm </td>
</tr>
<tr>
<td>Trypan Blue</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>for counting live/dead cells</td>
</tr>
<tr>
<td>BD Falcon Cell Strainer 40 um</td>
<td>Tool</td>
<td>BD Falcon</td>
<td>Falcon cat# 352340</td>
<td>Available through Fisher Scientific. Fisher catalog# 08-771-1</td>
</tr>
<tr>
<td>B27</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>17504-044</td>
<td>B27 is a proprietary supplement available through Invitrogen under their Gibco line of cell culture reagents.</td>
</tr>
<tr>
<td>Glutamax</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>35050-061</td>
<td>Glutamax is available through Invitrogen under their Gibco line of celll culture reagents.</td>
</tr>		</tbody>
		</table>
		</div>

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.

Watch this Scientific Journal Video about Preparing E18 Cortical Rat Neurons for Compartmentalization in a Microfluidic Device at JoVE.com

Preparing E18 Cortical Rat Neurons for Compartmentalization in a 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 ...

preparing-e18-cortical-rat-neurons-for-compartmentalization

Research

JoVE Journal

Biology

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Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, organotypic slice cultures allow one to perform a number of manipulations that are not presently feasible in-vivo. To date, organtotypic embryonic brain slice cultures have been used to follow individual cells using time-lapse microscopy, manipulate the expression of genes in the ganglionic emanances (a region that is hard to target by in-utero electroporation), as well as for pharmacological studies. In this video protocol we demonstrate how to make organotypic slice cultures from rat embryonic day 18 embryos. The protocol involves dissecting the embryos, embedding them on ice in low melt agarose, slicing the embedded brains on the vibratome, and finally plating the slices onto filters in culture dishes. This protocol is also applicable in its present form to making organotypic slice cultures from different embryonic ages for both rats and mice. 

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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.

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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.

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A Gradient-generating Microfluidic Device for Cell Biology

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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 ...

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Preparing T Cell Growth Factor from Rat Splenocytes

Maintenance of antigen-specific T cell lines or clones in culture requires rounds of antigen-induced activation separated by phases of cell expansion 1,2. Addition of interleukin 2 to the culture media during the expansion phase is necessary to prevent cell death and sufficient to maintain short-term T cell lines but has been shown to increase Th1 polarization 3. Replacement of interleukin 2 by T cell growth factor (TCGF) which contains a mix of cytokines is more effective than interleukin 2 in maintaining long-term T cell lines in vitro 3. Moreover, TCGF can easily be prepared in large amounts in the laboratory and is much cheaper than recombinant interleukin 2. 
Here, we show how to prepare TCGF from rat splenocyte culture supernatants. For this procedure, we harvest spleens from naive Lewis rats euthanized for thymus and blood collection. We prepare single-cell suspensions from the spleens, lyze the red blood cells by osmotic shock, and seed the splenocytes in culture medium. The cells are stimulated with concanavalin A, a mitogen that non-selectively activates all rat T lymphocytes, inducing the production of cytokines. The culture supernantant is collected 48 hours later andexcess concanavalin A is bound to alpha methyl mannoside to prevent it from activating T cell lines to which TCGF will be added. The TCGF is then sterile-filtered, aliquoted, and stored at -20&deg;C.

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Maintenance of antigen-specific T cell lines or clones in culture requires rounds of antigen-induced activation separated by phases of cell expansion 1,2. ...

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 ...

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Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia SCG

This is a protocol describing how to isolate and culture primary sympathetic neurons from superior cervical ganglia (SCG) of newborn rat pups.

The superior cervical ganglia (SCG) in rats are small, glossy, almond-shaped structures that contain sympathetic neurons.  These neurons provide ...

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Calcium imaging is a common technique that is useful for measuring calcium signals in cultured cells. Calcium imaging techniques take advantage of calcium indicator dyes, which are BAPTA-based organic molecules that change their spectral properties in response to the binding of Ca2+ ions. Calcium indicator dyes fall into two categories, ratio-metric dyes like Fura-2 and Indo-1 and single-wavelength dyes like Fluo-4. Ratio-metric dyes change either their excitation or their emission spectra in response to calcium, allowing the concentration of intracellular calcium to be determined from the ratio of fluorescence emission or excitation at distinct wavelengths.  The main advantage of using ratio-metric dyes over single wavelength probes is that the ratio signal is independent of the dye concentration, illumination intensity, and optical path length allowing the concentration of intracellular calcium to be determined independently of these artifacts. One of the most common calcium indicators is Fura-2, which has an emission peak at 505 nM and changes its excitation peak from 340 nm to 380 nm in response to calcium binding.  Here we describe the use of Fura-2 to measure intracellular calcium elevations in neurons and other excitable cells.

Calcium signals play a key role in many cellular processes including gene expression, survival and differentiation. Here we demonstrate how to perform calcium imaging using Fura-2 AM. Calcium imaging is a valuable tool to study the regulation of intracellular calcium in real time and its regulation of signaling cascades.

Calcium imaging is a common technique that is useful for measuring calcium signals in cultured cells. Calcium imaging techniques take advantage of calcium ...