This work was supported by NIH grant NS27501 to DKOD. Additional support for this work was provided by a grant to UC Irvine in support of DKOD through the HHMI Professor Program.

Preparations before day of culturing:
<ol>
<li>Make sterile dissecting solution.</li>
<li>Make sterile DMEM and keep in 10 ml aliquots at 4&deg;C for 2 weeks.</li>
<li>Make sterile DDM2 supplements and freeze in 50 or 100 &micro;l aliquots for 1 month.</li>
<li>Make ConA/laminin.</li>
<li>Coat coverslips. &nbsp;&nbsp;&nbsp;&nbsp; Optional: Make sterile CNBM and store frozen for up to 4 months. </li>
</ol>
On day of culturing
I.&nbsp; Prepare Enzyme Solution (ES) in laminar flow hood
<ol>
<li>Put 5 ml of dissecting solution (DS) in a 15 ml centrifuge tube.</li>
<li>Weigh 0.8 mg of L-Cysteine in a 0.6 ml eppendorf and add 150 &micro;l of DS. Pipette up and down until crystals are gone.</li>
<li>Add 150 ml of DS/Cysteine to the 5 ml DS and mix well.</li>
<li>Add 50 U (units) of Papain.</li>
<li>Add 7 ml of 0.1 N Na OH and mix well. Solution will clear within 30 min. when activated.&nbsp;</li>
<li>Filter enzyme solution with 0.2 mm syringe filter into a sterile 1.5 ml screw cap microfuge tube &nbsp; &nbsp;&nbsp; Papain: Want 50 U in 5 ml DS. Each batch is slightly different, so must calculate how much: &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 37.3 mg/ml and 28.3 U/mg &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 37.3 x 28.3 = 1055.59 U/ml&nbsp;&nbsp; (1000 ml) &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 50 U&nbsp; = 47.4 ml </li>
</ol>
II. Make DDM2 media
On day of culturing: add the following supplements to make DDM2
To 10 ml of DMEM add the supplements, just before culturing:
<ol>
<li>100&micro;l Transferrin</li>
<li>100&micro;l Putrescine</li>
<li>100&micro;l Selenium</li>
<li>100&micro;l Progesterone</li>
<li>50&micro;l&nbsp; Insulin</li>
<li>10&micro;l 20-Hydroxyecdysone</li>
</ol>
Note: Make enough DDM2 for all cultures planned (each brain plated on a single coverslip in an individual 35 mm Petri dish, 1.5 ms of media/culture)
Step III-VI can be at a done nonsterile lab bench and should be completed in 45 minutes or less.
III. Pupae Collection
<ol>
<li>Select 10-15 pupae from sides of vial under a dissecting microscope.</li>
<li>Place pupae in the lid of a dry 35 mm Petri dish.&nbsp;</li>
</ol>
Note: We generally use brains from pupae between 55-78 hours after puparium formation (APF). It is slightly harder to dissect out the brain from the younger pupae since the head capsules tend to collapse, while the overall level of neurite outgrowth is slightly lower from the oldest pupae. In the Canton-S wildtype strain, these stages are recognized by pigmented eyes that are light brown to slightly reddish, with little pigment elsewhere.
IV. Decapitation under dissecting microscope
<ol>
<li>Place two drops of sterile dissecting solution in lid of Petri dish, next to pupae.</li>
<li>Gently hold a single pupa with fine forceps in left hand and use a 27 gauge syringe needle attached to a 1 cc syringe in the right hand to remove the cuticle at the anterior end of pupa.</li>
<li>Squeeze pupa slightly with forceps and as head emerges use 27 gauge needle to sever neck.</li>
<li>With tip of the needle or the forceps, transfer head to drop of dissecting solution.</li>
<li>Repeat until you have 10-15 heads in a single drop.</li>
</ol>
V. Removal of the brain from head under dissecting microscope
<ol>
<li>In each hand hold a 1 cc syringe with a 27 gauge needle.</li>
<li>Use these to position the fly head in the drop of dissecting solution so the proboscis is facing you and the dorsal surface of the head is north.</li>
<li>Insert the left needle into the proboscis to pin the head down and use the right needle to make a slit in the right eye that goes from the ventral to the dorsal surface.</li>
<li>Insert the right needle near the left in the region of the proboscis and then use the left needle to gently depress the cuticle over the left eye, pushing the brain toward the slit in the right eye. The brain should emerge from the slit on the right, relatively intact, often with both optic lobes still attached and sometimes the pigmented eye tissue as well.</li>
<li>Push the brain onto the back of the needle and transfer to a drop of sterile dissecting saline in a new sterile, 35 mm Petri dish.</li>
<li>Collect 10-15 brains for each genotype. </li>
</ol>
VI. Removal of optic lobes and enzyme treatment
<ol>
<li>In each hand hold a 1 cc syringe with a 27 gauge needle and use these to gather all brains in one small area of the drop of dissecting solution</li>
<li>Use syringes as cutting tools to remove the optic lobes from each brain so the tissue remaining for culture is the central brain region.</li>
<li>Use a 20 &micro;l pipetman, set at 5 &micro;l, to transfer all the brains of a single genotype to an eppendorf tube containing 1 ml of sterile papain solution. </li>
<li>Brains incubated in papain for 10-15 minutes on a rotator set at 60 rpm.</li>
<li>Centrifuge at 3000 rpm for 3 minutes. </li>
</ol>
Inside sterile laminar flow hood &ndash; all steps where tissue is exposed to air should be done in laminar flow hood for the remaining steps.
VII. Washing
<ol>
<li>Remove enzyme solution and replace with sterile dissecting solution.</li>
<li>Centrifuge at 3000 rpm for 3 minutes.</li>
<li>Wash with sterile dissecting solution a total of 3 times.</li>
<li>Remove dissecting saline and replace with DDM2.</li>
<li>Centrifuge and wash a total of 2 times with DDM2.</li>
<li>Transfer brains and media to a sterile 35 mm Petri dish. </li>
</ol>
VIII. Trituration and Plating under dissecting microscope
<ol>
<li>Place 5 ul of DDM2 in the center of a coverslip.</li>
<li>Transfer 1 brain to this drop of DDM2 with a yellow tip.</li>
<li>Under the microscope, use 27 gauge needles to dice up brain into small pieces (should take &lt;1 minute).</li>
<li>Under visual guidance, break the tip of a&nbsp; glass pipet that has been pulled to a fine point so that the larger pieces can be gently sucked into the tip of the pipet and expelled back into the medium. We use mouth suction tubes that come standard with 100 &micro;l hematocrit capillary glass. Note: Take care not to get bubbles in the media and you will need to empirically determine the best size pipet to use. Good ones allow you to dissociate the brain with some individual cells and still some large clumps, in about 30 seconds/brain. When you look at these at higher power, approximately 10-20% of the neurons should still have some processes remaining and there will still be some large clumps. If you dissociate too much, then all the cells will be round and they will have sustained so much damage they will not survive. This step takes practice and must be done quickly since there is a large surface-to-volume ratio and the osmolality and pH of the drop of DDM2 can change quickly. Tissue needs to be placed in CO2 incubator as quickly as possible. </li>
<li>Write on the petri dish lid: "Genotype, date and time".</li>
<li>Put the 35 mm petri dish in a 100 mm petri dish (with wet kimwipe) and let settle in the CO2 incubator for 30 min.</li>
<li>Flood the 35 mm dish with 1.5 ml of DDM2.</li>
<li>Keep cultures in the incubator at 5% CO2 incubator (23&deg;C). Note: If you don&rsquo;t have access to a cooling CO2 incubator, use a standard mammalian tissue culture incubator and either put it in a cool room and heat to 23&deg;C, or put in the lab, turn off temp, and use ice in a tray at bottom of incubator to regulate temp around ambient. </li>
</ol>
IX. Feeding Cells
<ol>
<li>On Day 1 (next day), add 0.5 ml of CNBM/B27 (Conditioned Media) to make 3:1 DDM2:CNBM/B27.</li>
<li>On Day 5 replace 1 ml of media for 3:1 DDM2:CNBM/B27.</li>
<li>Keep feeding cells with 3:1 DDM2:CNBM/B27 every 4-5 days. Note: Cultures can be grown without the non-neuronal conditioned media but the neurons appear a bit more fragile and are more difficult for use in electrophysiology experiments.</li>
</ol>

<ol>
	<li>Banker, G., Goslin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culturing Nerve Cells. , (1991).</li><li>Rohrbough, J., O'Dowd, D. K., Baines, R. A., Broadie, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+bases+of+behavioral+plasticity:+Establishing+and+Modifying+synaptic+circuits+in+the+Drosophila+Genetic+System.">Cellular bases of behavioral plasticity: Establishing and Modifying synaptic circuits in the Drosophila Genetic System.</a> J. Neurobiol. 54, 254-271 (2003).</li><li>O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage-gated+currents+and+firing+properties+of+embryonic+Drosophila+neurons+grown+in+a+chemically+defined+medium.">Voltage-gated currents and firing properties of embryonic Drosophila neurons grown in a chemically defined medium.</a> J. Neurobiol. 27, 113-126 (1995).</li><li>Lee, D., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+excitatory+synaptic+transmission+mediated+by+nAChR+in+cultured+Drosophila+neurons.">Fast excitatory synaptic transmission mediated by nAChR in cultured Drosophila neurons.</a> J. Neurosci. 19, 5311-5321 (1999).</li><li>Su, H., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+synaptic+currents+in+Drosophila+mushroom+body+Kenyon+cells+are+mediated+by+alpha-bungarotoxin-sensitive+nAChRs+and+picrotoxin-sensitive+GABA+receptors.">Fast synaptic currents in Drosophila mushroom body Kenyon cells are mediated by alpha-bungarotoxin-sensitive nAChRs and picrotoxin-sensitive GABA receptors.</a> J. Neurosci. 23, 9246-9253 (2003).</li><li>Jiang, S. A., Campusano, J. M., Su, H., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+mushroom+body+Kenyon+cells+generate+spontaneous+calcium+transients+mediated+by+PLTX-sensitive+calcium+channels.">Drosophila mushroom body Kenyon cells generate spontaneous calcium transients mediated by PLTX-sensitive calcium channels.</a> J. Neurophysiol. 94, 491-500 (2005).</li><li>Campusano, J. M., Su, H., Jiang, S. A., Sicaeros, B., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=nAChR-mediated+calcium+responses+and+plasticity+in+Drosophila+Kenyon+cells.">nAChR-mediated calcium responses and plasticity in Drosophila Kenyon cells.</a> Develop Neurobiol. 67, 1520-1532 (2007).</li><li>Oh, H. -. W., Campusano, J. M., Hilgenberg, L. G. W., Sun, X., Smith, M. A., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+analysis+of+chemical+synapses+and+gap+junctions+between+Drosophila+brain+neurons+in+culture.">Ultrastructural analysis of chemical synapses and gap junctions between Drosophila brain neurons in culture.</a> Develop Neurobiol. , (2007).</li></ol>

Neurons harvested from the brains of embryonic/postnatal rodents can be grown in primary cell culture where they extend neurites and form functional synaptic connections. Methods for preparation of these cultures are well established and studies in rodent neuronal cultures have played a critical role in identifying genes and environmental factors involved in regulation of synapse formation and function (Banker and Goslin, 1991). While insect neurons from a variety of species can also be grown in culture, the only insect ...

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Concanavalin A</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C-2010</td>
<td>To 2.5 ml DS, add 25 mg Concanavalin A bottle. This is 10 mg/ml concentration.
Make aliquots of 90 ul and store at -20 C, no longer than 3 months.</td>
</tr>
<tr>
<td>Laminin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L-2020</td>
<td>Add 1ml DS to 1mg Laminin bottle. This is 0.5 mg/ml concentration.
Make aliquots of 10 ul and store at -20 C, no longer than 3 months.</td>
</tr>
<tr>
<td>Coverslips</td>
<td>&nbsp;</td>
<td>Bellco Biological Glassware</td>
<td>1943-00012</td>
<td>12 mm glass coverslips</td>
</tr>
<tr>
<td>ConA + Laminin solution</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Stock solution, from Con A stock and laminin stock, for coverslip coating: Add in 5 ml DS, 83.5 ul of ConA (167 ug/ml) and 8.35 ul of Laminin (0.835 ug/ml). Mix.
Make aliquots of 100 ul and store at -20 C, not longer than a month.</td>
</tr>
<tr>
<td>Dissecting Solution</td>
<td>Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>For 500 ml: 400 ml Ultra filtered water + 25 ml Stock Solution A + 14 ml Stock Solution B + 3.0 g (33.3 mM) D (+)-Glucose (Sigma G-8270) + 7.5 g (43.8 mM) Sucrose (Sigma S-0389). 

Adjust pH to 7.4 with 1N NaOH (around 2 ml). Bring final volume to 500 ml with ultra filtered water. Decant into a clean glass bottle and autoclave. Label "Dissecting Solution" and store at 4?C. </td>
</tr>
<tr>
<td>Dissecting Solution</td>
<td>Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>For 500 ml: 400 ml Ultra filtered water + 25 ml Stock Solution A + 14 ml Stock Solution B + 3.0 g (33.3 mM) D (+)-Glucose (Sigma G-8270) + 7.5 g (43.8 mM) Sucrose (Sigma S-0389). 

Adjust pH to 7.4 with 1N NaOH (around 2 ml). Bring final volume to 500 ml with ultra filtered water. Decant into a clean glass bottle and autoclave. Label "Dissecting Solution" and store at 4?C. </td>
</tr>
<tr>
<td>Solution B: HEPES</td>
<td>Buffer</td>
<td>Sigma</td>
<td>H-3375</td>
<td>20.97g (9.9mM). 

Add ultra filtered water up to 200 ml. Mix until dissolve and bring final volume to 250 ml. Place in a clean bottle and autoclave. Label "Solution B" and store at 4 C. </td>
</tr>
<tr>
<td>Solution A</td>
<td>Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>For 500 ml: 80.0g (137 mM) NaCl (Sigma S-9625) + 4.0g (5.4mM) KCl (Sigma P-4504) + 0.24g (0.17mM) Na2HPO4 (Sigma S-0876) + 0.3g (0.22 mM) KH2PO4 (Sigma P-5379).

Weigh out all ingredients and mix until dissolved with 400 ml ultra filtered water. Bring final volume to 500 ml. Place in a clean bottle and autoclave. Label "Solution A" and store at 4&#176;C. 

</td>
</tr>		</tbody>
		</table>
		</div>
			<div>
			Coating Coverslips:

Put autoclaved coverslips in a 60 mm petri dish.
Pipet 5 ul of Con A/Laminin mix onto center of each coverslip.
Place in 37 C incubator for 2 hours.
Rinse 3x coverslips with 100 ul of autoclaved water each. Use vacuum attached to a 
sterile Pasteur pipet.
During the last rinse, pick up the coverslip with forceps and dry both sides.
Transfer the coverslip to a 35mm Petri dish.
Store at room temperature for up to a month.		</div>

primary neuronal cultures from the brains of late stage drosophila pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as  GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.

Watch this Scientific Journal Video about Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae at JoVE.com

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2.

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

primary-neuronal-cultures-from-brains-late-stage-drosophila

Research

JoVE Journal

Biology

15.0K 조회수.  University of California, Irvine (UCI). 이 동영상은 늦게 무대 Drosophila pupae의 두뇌에서 기본의 연결을 문화의 준비를 보여줍니다. 라이브 문화의 전망 Fura - 2를 사용하여 칼슘 수준의 neurite의 파생물 및 이미징을 보여줍니다.

비디오: Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information into directional movement towards the appropriate target cell. To fully understand how guidance information is transmitted from the cell surface to the underlying dynamic cytoskeletal networks, one needs a model system suitable for live cell imaging of protein dynamics at high temporal and spatial resolution. Typical vertebrate growth cones are too small to quantitatively analyze F-actin and microtubule dynamics. Neurons from the sea hare Aplysia californica are 5-10 times larger than vertebrate neurons, can easily be kept at room temperature and are very robust cells for micromanipulation and biophysical measurements. Their growth cones have very defined cytoplasmic regions and a well-described cytoskeletal system. The neuronal cell bodies can be microinjected with a variety of probes for studying growth cone motility and guidance. In the present protocol we demonstrate a procedure for dissection of the abdominal ganglion, culture of bag cell neurons and setting up an imaging chamber for live cell imaging of growth cones.

Aplysia californica neurons develop large growth cones in culture that are excellent for high-resolution imaging of growth cone motility and guidance. Here, we present a protocol for dissection and plating of Aplysia bag cell neurons as well as for setting up a chamber for live cell imaging.

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information ...

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons. The neurons are large (up to 1 mm in diameter) and identifiable, with distinct sizes, shapes, positions and pigmentations, and the cell bodies are externally exposed in five paired ganglia distributed throughout the body of the animal. These properties have allowed investigators to delineate the circuitry underlying specific behaviors in the animal1. The monosynaptic connection between sensory and motor neurons is a central component of the gill-withdrawal reflex in the animal, a simple defensive reflex in which the animal withdraws its gill in response to tactile stimulation of the siphon. This reflex undergoes forms of non-associative and associative learning, including sensitization, habituation and classical conditioning. Of particular benefit to the study of synaptic plasticity, the sensory-motor synapse can be reconstituted in culture, where well-characterized stimuli elicit forms of plasticity that have direct correlates in the behavior of the animal2,3. Specifically, application of serotonin produces a synaptic strengthening that, depending on the application protocol, lasts for minutes (short-term facilitation), hours (intermediate-term facilitation) or days (long-term facilitation). In contrast, application of the peptide transmitter FMRFamide produces a synaptic weakening or depression that, depending on the application protocol, can last from minutes to days (long-term depression). The large size of the neurons allows for repeated sharp electrode recording of synaptic strength over periods of days together with microinjection of expression vectors, siRNAs and other compounds to target specific signaling cascades and molecules and thereby identify the molecular and cell biological steps that underlie the changes in synaptic efficacy.
An additional advantage of the Aplysia culture system comes from the fact that the neurons demonstrate synapse-specificity in culture4,5. Thus, sensory neurons do not form synapses with themselves (autapses) or with other sensory neurons, nor do they form synapses with non-target identified motor neurons in culture. The varicosities, sites of synaptic contact between sensory and motor neurons, are large enough (2-7 microns in diameter) to allow synapse formation (as well as changes in synaptic morphology) with target motor neurons to be studied at the light microscopic level.
In this video, we demonstrate each step of preparing sensory-motor neuron cultures, including anesthetizing adult and juvenile Aplysia, dissecting their ganglia, protease digestion of the ganglia, removal of the connective tissue by microdissection, identification of both sensory and motor neurons and removal of each cell type by microdissection, plating of the motor neuron, addition of the sensory neuron and manipulation of the sensory neurite to form contact with the cultured motor neuron.

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

Primary cultures of Aplysia sensory-motor neurons provide a model preparation for studying synapse formation and synaptic plasticity in vitro. This video demonstrates the identification and microdissection of sensory and motor neurons from Aplysia ganglia as well as the methods for establishing and maintaining sensory-motor neurons in culture.

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons.  The neurons are large (up ...

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three preparations of the brain and visual system that cover the range from the developing eye disc-brain complex in the developing pupae to individual eye and brain dissection from adult flies. All protocols are optimized for the live culture of the preparations. However, we also present the conditions for fixed tissue immunohistochemistry where applicable. Finally, we show live imaging conditions for these preparations using conventional and resonant 4D confocal live imaging in a perfusion chamber. Together, these protocols provide a basis for live imaging on different time scales ranging from functional intracellular assays on the scale of minutes to developmental or degenerative processes on the scale of many hours.

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three ...

Establishing Primary Adult Fibroblast Cultures From Rodents

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized. Primary cells are preferred in studies of cell cycle control, apoptosis, and DNA repair, as cancer cells carry mutations in genes involved in these processes. Primary cells cannot be cultured indefinitely due to the onset of replicative senescence or aneuploidization. Hence, new cultures need to be established regularly. The procedure for isolation of rodent embryonic fibroblasts is well established, but isolating adult fibroblast cultures often presents a challenge. Adult rodent fibroblasts isolated from mouse models of human disease may be a preferred control when comparing them to fibroblasts from human patients. Furthermore, adult fibroblasts are the only available material when working with wild rodents where pregnant females cannot be easily obtained. Here we provide a protocol for isolation and culture of adult fibroblasts from rodent skin and lungs. We used this procedure successfully to isolate fibroblasts from over twenty rodent species from laboratory mice and rats to wild rodents such as beaver, porcupine, and squirrel.

This article describes a protocol for isolation and maintenance of primary fibroblast cultures from skin and lung tissue of wild rodents.

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized.  Primary cells are preferred ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&amp;#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This methods paper describes how to isolate mid-to-late stage follicles (Stage 10B-14) and utilize them to provide new insights into the molecular and morphologic events occurring during tight windows of developmental time. Isolated follicles can be used for a variety of experimental techniques, including in vitro development assays, live imaging, mRNA expression analysis and western blot analysis of proteins. Follicles at Stage 10B (S10B) or later will complete development in culture; this allows one to combine genetic or pharmacologic perturbations with in vitro development to define the effects of such manipulations on the processes occurring during specific periods of development. Additionally, because these follicles develop in culture, they are ideally suited for live imaging studies, which often reveal new mechanisms that mediate morphological events. Isolated follicles can also be used for molecular analyses. For example, changes in gene expression that result from genetic perturbations can be defined for specific developmental windows. Additionally, protein level, stability, and/or posttranslational modification state during a particular stage of follicle development can be examined through western blot analyses. Thus, stage-specific isolation of Drosophila follicles provides a rich source of information into widely conserved processes of development and morphogenesis.

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

Stage-specific isolation of mid-to-late Drosophila follicles is useful for a variety of purposes. Such follicles develop in culture, which allows for genetic and/or pharmacologic manipulations to be coupled with in vitro development assays and live imaging. Additionally, follicles can be used for molecular studies, such as isolating mRNA and protein.

Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This ...