Preparations before day of culturing:
<ul>
<li>Prepare sterile dissecting solution (DS).</li>
<li>Prepare NBM/B27 (Neurobasal Mediom with B27 supplements).</li>
<li>Autoclave ddH2O and sterilize glass coversilps, if needed.</li>
<li>Coat tissue culture dishes or glass coverslips with poly-D-lysine.</li>
</ul>
Poly-D-Lysine Coating:
Prepare the day before culturing under sterile conditions.
<ul>
<li>Thaw aliquot of 10X PDL and place on ice.</li>
<li>Add 9 ml sterile ultra-filtered water to 1 ml of PDL and mix well (1X).</li>
<li>Coat surfaces with 1X PDL overnight at room temperature as follows: 
<ul>
<li>glass coverslips (Bellco Glass, Inc. Cat.# 1943-00012): 75 &micro;l/coverslip &nbsp;OR</li>
<li>24 well culture plate: 300 &micro;l/well &nbsp;OR</li>
<li>35mm culture dish: 1ml/dish</li>
</ul>
</li>
<li>Rinse 5X with sterile water (use sterile Pasteur pipettes to aspirate liquid).</li>
<li>Remove water until surface is completely dry.</li>
</ul>
Culturing procedure (done in laminar flow hood under sterile conditions)
<ol>
<li>Cut out block of agar and glue it onto the support block of the microtome Vibraslicer (Campden Instruments Ltd.) using super glue.</li>
<li>When using late embryonic stage (E17-18) mouse fetuses, euthanise dam, remove uterus and free individual fetuses from embryonic sack. Place fetuses into sterile Petri dish and continue as outlined below.</li>
<li>Decapitate mouse fetus or pup (follow guidelines approved by your Institutional Animal Care and Use Committee).</li>
<li>Remove skin and skull and place brain onto Whatman filter paper disk in a 60mm petri dish filled with cold DS.</li>
<li>Cut off the cerebellum using a sterile razor blade.</li>
<li>Pick up brain using a spatula and drain excess fluid on filter paper, then transfer brain to Vibraslicer support block and glue brain in place (caudal side up and ventral part facing agar). </li>
<li>Fill chamber with cold DS. Set speed selector to 8-9.</li>
<li>Cut 200-400 &micro;m sections, beginning from olfactory bulb. Once the blade of the Vibraslicer enters the cortex, begin cutting 600&micro;m coronal sections. An E17-18 mouse brain typically yields 3-4 usable slices, while a P0 mouse brain yields 4-5 usable slices.</li>
<li>Transfer brain slices to 35 mm petri dish labeled DS 2 using reverse end of un-plugged 10 ml glass Pasteur pipet.</li>
<li>Dissect out cortex and remove meninges using glass needles pulled from capillary tubes.</li>
<li>Cut cortical rinds into small pieces, 0.5-1 mm in length.</li>
<li>Filter ES through 0.2 &micro;m filter attached to a 5 cc syringe into a 35 mm petri dish.</li>
<li>Transfer the cortical tissue pieces to petri dish containing filtered ES.</li>
<li>Incubate at 37&deg;C for 30 min.</li>
</ol>
In the meantime:
<ol>
<li>Clean up hood, Vibraslicer and dissecting tools. </li>
<li>Add 5 ml of warm NBM/B27 into a 60 mm petri dish. </li>
</ol>
After 30 min. incubation:
<ol>
<li>Transfer tissue to 10 ml DS. Let settle for 1 min.</li>
<li>Transfer tissue to first Hi tube swirls and let settle for 2-3 min.</li>
<li>Transfer to second Hi. Repeat as above.</li>
<li>Transfer to first Li. Repeat as above.</li>
<li>Transfer to second Li. Repeat as above.</li>
<li>Transfer to third Li. Repeat as above.</li>
<li>Transfer tissue to the 60 mm dish with media.</li>
</ol>
Under the dissection microscope:
<ol>
<li>Clean debris from tissue and triturate each piece by gently passing through a pulled glass pipet (use decreasing bore sizes) to loosen up the tissue.</li>
<li>Transfer cell suspension to 15 ml conical tube containing the rest of NBM+B27 (Make a total of 13 ml for a 24 well plate or 11 ml for 5 - 35 mm dishes. Mix gently and wait for large pieces of tissue to settle).</li>
<li>Transfer cell suspension (0.5 ml/well for a 24 well plate or 2 ml / 35 mm culture dish). </li>
<li>When using glass coverslips, add 80 &micro;l cell suspension per coverslip and allow 1 hr in tissue culture incubator for cells to adhere before flooding the chamber with more NBM/B27 media.</li>
<li>Maintain cultures at 37&deg;C and 5% CO2.</li>
</ol>
Next Day
24 well plate: Feed each well with 0.5 ml non-neuronal cell conditioned NBM/B27 medium (cNBM/B27; Protocol for preparation of cNBM/B27 medium appears below)
35mm dishes: Replace 0.5 ml with cNBM/B27 medium.
Maintain culture by replacing 0.5ml of medium with fresh cNBM/B27 every 2-3 days.
Although the culture media does not promote glia cell proliferation, cultures can be treated with 5&micro;M FDU (5-Fluoro-2&rsquo;-deoxyuridine, Sigma F0503) at day 3-5 in culture to further reduce the number of glial cells if needed.
SET-UP for Dissection and Culture
Sterilize dissecting tools in 70% EtOH or autoclave them:
<ul>
<li>Scissors (med. and small) Spatulas (med. and small)</li>
<li>Tweezers (med. and small) Razor blade</li>
<li>Blade for vibraslicer Buffer bath</li>
<li>Small metal wedge for blade Support for brain</li>
</ul>
Other materials:
<ul>
<li>Petri dishes (60 mm)</li>
<li>Petri dishes (35 mm)</li>
<li>Filter paper</li>
<li>Glass pipet 10 ml</li>
<li>Sterile disposable pipets, 5,10 and 25 ml</li>
<li>Syringe filter, 0.2 &micro;m</li>
<li>Syringe 5 cc</li>
<li>Centrifuge tubes, 15 ml</li>
<li>Sterile Pasteur glass pipets</li>
<li>PDL coated culture dishes or glass coverslips</li>
</ul>
Label six 15 ml centrifuge tubes and follow preparation:
Tube #1: Enzyme solution:
Add 50 U of papain (Worthington LS 03126) to 5 ml DS containing:
<ul>
<li>100&micro;l of L-cystein (0.8mg)</li>
<li>7 &micro;l 0.1N NaOH</li>
<li>50 &micro;l APV (5mM)</li>
</ul>
Leave solution out at room temperature to clear. Note that enzyme solution will appear &lsquo;cloudy&rsquo; at first and needs to clear before use.
&nbsp;
Tube #2 &amp; #3: Hi Enzyme Inhibitor:
3 ml DS + 300 &micro;l BSA/Ti + 30 &micro;l APV (5mM).
Mix gently to avoid bubbles, then divide into two 1.5ml aliquots.
&nbsp;
Tube #4 - #6: Low Enzyme Inhibitor:
8 ml DS + 80 &micro;l BSA/Ti + 80 &micro;l APV (5mM).
Mix gently to avoid bubbles, divide into three 2.6 ml aliquots.
&nbsp;
Place tubes numbered #2 through #6 on ice. Leave tube #1 (Enzyme Solution) at room temperature.
SOLUTIONS AND ALIQUOTS
<ul>
<li>Solution A (Buffered Saline) Sigma Formula wt 500 ml [conc.]</li>
<li>Sodium Chloride NaCl S-9625 58.45 80.0g 137mM</li>
<li>Potassium Chloride KCl P-4504 74.56 4.0g 5.4mM</li>
<li>Sodium Phosphate Dibasic anhydrous Na2HPO4 S-0876 142.0 0.24g 0.17mM</li>
<li>Potassium Phosphate Monobasic anhydrous KH2PO4 P-5379 136.09 0.3g 0.22mM</li>
</ul>
Weigh out all ingredients and mix until dissolve with 400 ml ultra filtered water. Bring final volume to 500 ml. Place in a clean bottle and autoclave. Store at 4&deg;C and label "DS Solution A".
<ul>
<li>Solution B (Hepes) Sigma Formula wt 250 ml [conc.]</li>
<li>Hepes Hepes H-3375 283.3 20.97g 9.9mM</li>
</ul>
Add ultra-filtered water up to 200 ml. Mix until dissolved and bring final volume to 250 ml. Place in a clean bottle and autoclave. Store at 4&deg;C and label "DS Solution B".
<ul>
<li>Working Solution 500 ml [conc.]</li>
<li>Ultra filtered water 400 ml </li>
<li>Stock solution A 25 ml </li>
<li>Stock Solution B 14 ml </li>
<li>D (+)-Glucose Sigma G-8270 3.0 g 33.3mM</li>
<li>Sucrose Sigma S-0389 7.5 g 43.8 mM</li>
</ul>
Adjust pH to 7.4 with 1N NaOH. Bring final volume to 500 ml with ultra filtered water. Decant into a clean glass bottle and autoclave. Store at 4&deg;C. Label "Dissecting Solution".
Poly-D-Lysine Preparation:
<ol>
<li>Prepare 10x stock solution of poly-D-lysine (PDL; Sigma P-7280) in sterile H2O at 1mg/ml.</li>
<li>Make 1.0 ml aliquots. This solution can be stored at -20&deg;C for up to 3 months.</li>
</ol>
MEDIA
<ol>
<li>Add 10ml of B-27 Supplement (Gibco 17504-044) to 500 ml NBM bottle (Neurobasal Medium, Gibco 21103-049).</li>
<li>Make 40 ml aliquots and store at 4&deg;C.</li>
</ol>
APV
<ol>
<li>Add 10 ml sterile H2O to vial of 10 mg APV (2-Amino-5-phosphonopentanoic acid, Sigma A-5282) and mix thoroughly.</li>
<li>Prepare 180 &micro;l aliquots and store at -20&deg;C.</li>
</ol>
BSA/Ti
<ol>
<li>Dissolve 1 g BSA (Bovine Albumin. Sigma A-7030) and 1 g Trypsin Inhibitor (Sigma T-9253) in 10 ml DS.</li>
<li>Adjust pH to 7.4 with 1N NaOH.</li>
<li>Sterilize by filtering through 0.2 &micro;m syringe filter.</li>
<li>Divide into 400 &micro;l aliquots and store at -20&deg;C.</li>
</ol>
L-CYSTEINE
In an Eppendorf tube dissolve 0.6 mg L-Cysteine (Sigma C-7755) in 200 &micro;l DS.
AGAR (4%)
<ol>
<li>Dissolve 4 g of Bacto Agar (Difco 0140-01) in 100 ml sterile water. Keep at 4&deg;C until needed for culture.</li>
<li>Microwave Agar to melt and fill a 35 mm petri dish. Let sit to cool down and polymerize.</li>
</ol>
PAPAIN (Worthington LS 03126)
NON-NEURONAL CULTURES IN NUNC CULTURE BOTTLES
COATING CULTURE BOTTLE (4 Nunc bottles)
<ol>
<li>Add 9 mL sterile water to each of 3 tubes of 10X PDL to make 1X PDL.</li>
<li>Transfer 30 ml 1X PDL in the first bottle.</li>
<li>Move slightly until all the growth surfaces are covered. Let sit for 1 min.</li>
<li>Transfer 1x PDL to second bottle. Let sit for 1 min. </li>
<li>Repeat with the third and forth bottles.</li>
<li>Rinse all the four bottles 2X with 150 ml sterile H2O.</li>
</ol>
PREPARE ENZYME SOLUTION (ES):
<ol>
<li>In a 0.6 ml centrifuge tube weight out 0.8 mg L-Cysteine (Sigma C7755), add 150ml DS and vortex until crystals are dissolved.</li>
<li>Transfer 5 ml DS to a 15 ml conical tube, then add 150ml L-Cysteine.</li>
<li>Add 50 units Papain. (Worthington LS 03126)</li>
<li>Add 7ml 0.1N NaOH.</li>
</ol>
PREPARE MEM (GIBCO # 11090-081)
<ol>
<li>Remove 65 ml from the 500 ml MEM bottle. Save 10 ml to prepare Glucose.</li>
<li>Add 5 ml Pen/Strep (Gibco #15070-063).</li>
<li>Add 10 ml 1M Glucose (Sigma G-8270) (1.8 g/ 10 ml MEM)</li>
<li>Add 50 ml Fetal Bovine Serum (Gibco # 16140-071) or Bovine Calf Serum (Omega BC-04).</li>
<li>Mix well, aliquot and store at 4&deg;C.</li>
</ol>
NEUROBASAL MEDIUM/B-27 SUPPLEMENT (NBM/B27) NBM, 500 ml (Gibco #21103-049); B-27 (50X), 10 ml (Gibco #17504-044)
<ol>
<li>Add the entire content of B-27 vial to the NBM bottle and mix. </li>
<li>Aliquot and store at 4&deg;C. </li>
</ol>
DISSECTION OF BRAIN TISSUE
<ol>
<li>Dry dissecting tools from 70% EtOH before dissection.</li>
<li>Transfer 10 ml DS in each of 3-15 ml tube.</li>
<li>Select mouse pups (P0 &ndash; P3), you will need three brains for 4 bottles.</li>
<li>Add cold DS to 35 mm petri dish.</li>
<li>Decapitate mouse pup and dissect out brains.</li>
<li>Place brains into the petri dish containing cold DS.</li>
<li>Chop tissue into pieces, small enough to pass through a glass pipette.</li>
</ol>
ENZYME DISSOCIATION
<ol>
<li>Remove as much DS as possible from the dish with a Pasteur pipette.</li>
<li>Filter ES through a 0.2 &micro;m filter and 5cc syringe.</li>
<li>Place ES in the dish with tissue.</li>
<li>Incubate tissue at 37&deg;C incubator (CO2 free) for 30 min.</li>
</ol>
WASHING TISSUE
<ol>
<li>Transfer tissue with glass pipette to the first DS tube, making sure to get as little ES as possible.</li>
<li>Centrifuge for 30 sec at 1500 rpm.</li>
<li>Transfer tissue and repeat procedure two more times.</li>
</ol>
TRITURATION
<ol>
<li>Place 38 ml of MEM/FBS into a 50ml centrifuge tube</li>
<li>Add 3 ml of MEM/FBS to a 35 mm dish and transfer tissue to this petri dish.</li>
<li>Dissociate the tissue by gently triturating it through a 1000&micro;l eppendorf pipet tip.</li>
<li>Transfer cell suspension to the tube containing 38ml of MEM/FBS and mix well.</li>
</ol>
PLATING
<ol>
<li>Stand the culture bottles upright to get equal amounts of the cell suspension and media in each bottle.</li>
<li>Put 90 ml of MEM/FBS in each culture bottle.</li>
<li>Add 10 ml of cell suspension in each bottle.</li>
<li>Label: Non-Neuronal, initials and date. Incubate at 37&deg;C and 5% CO2.</li>
</ol>
FEEDING
<ol>
<li>On Day 3 or 4, feed cells with warm MEM/FBS (decant all the media and replace it with 100 ml of MEM/FBS). Cultures may take 7-10 days to become confluent.</li>
<li>On Day 7-10 change the media for NBM/B27.</li>
<li>Next day, collect the media. Filter with 0.22&micro;m </li>
<li>Make aliquots of 40 ml and feed culture with MEM/FBS.</li>
<li>Next 2 or 3 days, change media for NBM/B27</li>
<li>Next day, collect&nbsp;and repeat the procedure.</li>
<li>Keep collecting media for approx. 2 weeks.</li>
</ol>

The authors have nothing to disclose.

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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>5-Fluoro-2'-deoxyuridine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F0503</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Chloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S9625</td>
<td>&nbsp;</td>
<tr>
<td>Vibraslicer</td>
<td>&nbsp;</td>
<td>Campden Instruments Ltd.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Potassim Chloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P4504</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Phosphate Dibasic</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S0876</td>
<td>&nbsp;</td>
<tr>
<td>Potassium Phosphate Monobasic</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P5379</td>
<td>&nbsp;</td>
<tr>
<td>Hepes</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H3375</td>
<td>&nbsp;</td>
<tr>
<td>D (+)-Glucose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G8270</td>
<td>&nbsp;</td>
<tr>
<td>Sucrose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S0389</td>
<td>&nbsp;</td>
<tr>
<td>Poly-D-Lysine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P7280</td>
<td>&nbsp;</td>
<tr>
<td>B27 Supplement</td>
<td>&nbsp;</td>
<td>Invitrogen (Gibco)</td>
<td>17504-044</td>
<td>&nbsp;</td>
<tr>
<td>Neurobasal Media (NBM)</td>
<td>&nbsp;</td>
<td>Invirtogen (Gibco)</td>
<td>21103-049</td>
<td>&nbsp;</td>
<tr>
<td>2-Amino-5-phosphonopentanoic acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A5282</td>
<td>&nbsp;</td>
<tr>
<td>Bovine Albumin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A7030</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin Inhibitor</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T9253</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Hydroxide, 1N solution</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>SS266-1</td>
<td>&nbsp;</td>
<tr>
<td>L-Cysteine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C7755</td>
<td>&nbsp;</td>
<tr>
<td>Bacto Agar</td>
<td>&nbsp;</td>
<td>Difco</td>
<td>0140-01</td>
<td>&nbsp;</td>
<tr>
<td>Papain</td>
<td>&nbsp;</td>
<td>Worthington </td>
<td>LS 03126</td>
<td>&nbsp;</td>
<tr>
<td>Sterile 0.2 &micro;m Syringe Filter</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>DDA02025S0</td>
<td>&nbsp;</td>
<tr>
<td>Glass Coverslips, No1, &#216;12mm</td>
<td>&nbsp;</td>
<td>Bellco Glass Inc.</td>
<td>1943-00012</td>
<td>&nbsp;</td>
<tr>
<td>Minimum Essential Media (MEM)</td>
<td>&nbsp;</td>
<td>Invitrogen (Gibco)</td>
<td>11090-081</td>
<td>with Earle's salts
without L-glutamine, needed for growing non-neuronal cultures</td>
</tr>
<tr>
<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen (Gibco)</td>
<td>15070-063</td>
<td>for non-neuronal culture</td>
</tr>
<tr>
<td>Fetal Bovine Serum</td>
<td>&nbsp;</td>
<td>Invitrogen (Gibco)</td>
<td>16140-071</td>
<td>needed for non-neuronal cultures</td>
</tr>
<tr>
<td>Nunclon "Triple Flask" Tissue Culture Bottle</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>12-565-25</td>
<td>Use for non-neuronal cultures. these flasks are very convenient when producing "conditioned Neurobasal Media/B27", but any other tissue culture flasks or dishes can be used instead.</td>
</tr>
<tr>
<td>Glass bottom "Imaging dishes"</td>
<td>&nbsp;</td>
<td>MatTek Corp.</td>
<td>P35G-1.5-10-C</td>
<td>Glass bottomed, &#216;35mm culture dishes ideal for Calcium or Sodium Imaging when using an inverted imaging setup. But expensive!</td>
</tr>		</tbody>
		</table>
		</div>

preparation of dissociated mouse cortical neuron cultures

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures can be used for a variety of applications including immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging, protein and/or RNA isolation. These cultures also provide a platform to study the neuronal development of transgenic animals that carry a late embryonic or postnatal lethal gene mutation. The procedure is relatively straight forward, requires some experience in tissue culture technique and should not take longer than two to three hours if you are properly prepared. Careful separation of the cortical rind from the thalamo-cortical fiber tract will reduce the number of unwanted non-neuronal cells. To increase yields of neuronal cells triturate the pieces of the cortical tissue gently after the enzyme incubation step. This is imperative as it prevents unnecessary injury to cells and premature neuronal cell death. Since these cultures are maintained in the absence of glia feeder cells, they also offer an added advantage of growing cultures enriched in neurons.

Watch this Scientific Journal Video about Preparation of Dissociated Mouse Cortical Neuron Cultures at JoVE.com

Preparation of Dissociated Mouse Cortical Neuron Cultures

This video shows a procedure for generating neuronal cultures from late embryo and early postnatal mouse cortex. These cultures can be used for immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging and provide a platform to study the neuronal development of transgenic animals that carry a postnatal lethal gene mutation.

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures ...

preparation-of-dissociated-mouse-cortical-neuron-cultures

Research

JoVE Journal

Biology

43.3K vistas.  University of California, Irvine (UCI). Este video muestra un procedimiento para la generación de cultivos neuronales a partir de finales de embriones y la corteza temprana de ratón postnatal. Estos cultivos pueden ser utilizados para inmunocitoquímica, la bioquímica, la electrofisiología, calcio y sodio de imagen y proporcionar una plataforma para estudiar el desarrollo neuronal de los animales transgénicos que portan una mutación del gen letal después del parto.

Video: Preparation of Dissociated Mouse Cortical Neuron Cultures

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 ...

Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the physiological property to generate a wide array of differentiated cell types, including neurons, nerve supporting cells, smooth muscle cells, bone/cartilage cells and melanocytes. EPI-NCSC are easily accessible in the hairy skin and can be isolated as a highly pure population of stem cells. This video provides a detailed protocol for preparing mouse EPI-NCSC cultures from whisker follicles. The whisker pad of an adult mouse is removed, and whisker follicles dissected. The follicles are then cut longitudinally and subsequently transversely above and below the bulge region. The bulge is removed from the collagen capsule and placed in a culture plate. EPI-NCSC start to emigrate from the bulge explants 3 to 4 days later.

Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

Here we show our method to isolate mouse epidermal neural crest stem cells (EPI-NCSC). Technique involves micro-dissecting whisker follicles, isolating the bulge and placeing it into tissue culture. EPI-NCSC start to emigrate from bulge explants onto the substratum within 3 - 4 days.

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the ...

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 ...

Preparation of 2-dGuo-Treated Thymus Organ Cultures

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play a key role in the development of a functionally competent T-cell pool. However, the complexity of T-cell development in the thymus in vivo can limit analysis of individual cellular components and particular stages of development. In vitro culture systems provide a readily accessible means to study multiple complex cellular processes. Thymus organ culture systems represent a widely used approach to study intrathymic development of T-cells under defined conditions in vitro. Here we describe a system in which mouse embryonic thymus lobes can be depleted of endogenous haemopoeitic elements by prior organ culture in 2-deoxyguanosine, a compound that is selectively toxic to haemopoeitic cells. As well as providing a readily accessible source of thymic stromal cells to investigate the role of thymic microenvironments in the development and selection of T-cells, this technique also underpins further experimental approaches that include the reconstitution of alymphoid thymus lobes in vitro with defined haemopoietic elements, the transplantation of alymphoid thymuses into recipient mice, and the formation of reaggregate thymus organ cultures. (This article is based on work first reported Methods in Molecular Biology 2007, Vol. 380 pages 185-196).

This video demonstrates the dissection and removal of the fetal thymus as well the preparation of ex vivo cultures of 2-dGuo-treated thymus.

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play ...

Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for neuronal survival and function. Neurotrophins are specifically required to maintain the survival and differentiation of innervating sensory neurons but the question of how these target-derived neurotrophins communicate to the cell body of innervating neurons has been an area of active research for over 30 years. The most commonly accepted model of how neurotrophin signals reach the cell body proposes that signaling endosomes carry this signal retrogradely along the axon. In order to study retrograde transport, a culture system was originally devised by Robert Campenot, in which cell bodies are isolated from their axons. The technique of preparing these compartmented chambers for culturing sensory neurons recapitulates the selective stimulation of neuron terminals that occurs in vivo following release of target-derived neurotrophins. Retrograde signaling events that require long-range microtubule dependent retrograde transport have important implications for the treatment of neurodegenerative disorders.

Here we describe the technique of preparing and maintaining compartmented chambers for culturing sensory neurons of the dorsal root ganglia.

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for ...

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons 

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the cerebellar neuronal types (granule cells, Purkinje cells and inhibitory interneurons), granule neurons are by far the most numerous and are the most abundant type of neurons in the mammalian brain. In rodents, cerebellar granule neurons are generated during the first two post-natal weeks from progenitor cells in the outermost layer of the cerebellar cortex, the external granule layer (EGL). The protocol presented here describes techniques to enrich and culture granule neurons and their progenitor cells from post-natal mouse cerebellum. We will describe procedures to obtain cultures of increasing purity3,4, which can be used to study the differentiation of proliferating progenitor cells into granule neurons5,6. Once the progenitor cells differentiate, the cultures also provide a homogenous population of granule neurons for experimental manipulation and characterization of phenomena such as synaptogenesis, glutamate receptor function7, interaction with other purified cerebellar cells8,9 or cell death7.

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons

Here we present a method to isolate and culture cerebellar granule neuron progenitor cells and cerebellar granule neurons from postnatal mouse.

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the ...

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 Rat Brain Aggregate Cultures for Neuron and Glia Development Studies

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) would provide a powerful platform for neuroscientists to investigate axo-glial interactions, properties and differentiation of multipotent progenitors, and progression of oligodendroglial lineage cells at the cellular and molecular level.  We describe here a CNS aggregate culture system from embryonic rat forebrains, which can be maintained in a serum-free medium up to 3-4 weeks and is used in our laboratory as a model to study neuron-glia interaction and CNS myelination.  This video clip will demonstrate how to isolate and grow these CNS aggregate cultures from E16 rat brain.  Furthermore, from the same brain dissection, highly enriched regular dissociated neuronal cultures can be readily obtained and used for various studies on CNS neurons or used for co-cultures with other cells. 

A protocols for an embryonic rat brain aggregate culture system is described. Multipotent progenitors in the aggregates can develop and differentiate into neurons, astrocytes and oligodendrocytes.

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) ...

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 ...

Intranuclear Microinjection of DNA into Dissociated Adult Mammalian Neurons

Primary neuronal cell cultures are valuable tools to study protein function since they represent a more biologically relevant system compared to immortalized cell lines. However, the post-mitotic nature of primary neurons prevents effective heterologous protein expression using common procedures such as electroporation or chemically-mediated transfection. Thus, other techniques must be employed in order to effectively express proteins in these non-dividing cells.
In this article, we describe the steps required to perform intranuclear injections of cDNA constructs into dissociated adult sympathetic neurons. This technique, which has been applied to different types of neurons, can successfully induce heterologous protein expression. The equipment essential for the microinjection procedure includes an inverted microscope to visualize cells, a glass injection pipet filled with cDNA solution that is connected to a N2(g) pressure delivery system, and a micromanipulator. The micromanipulator coordinates the injection motion of microinjection pipet with a brief pulse of pressurized N2 to eject cDNA solution from the pipet tip. 
This technique does not have the toxicity associated with many other transfection methods and enables multiple DNA constructs to be expressed at a consistent ratio. The low number of injected cells makes the microinjection procedure well suited for single cell studies such as electrophysiological recordings and optical imaging, but may not be ideal for biochemical assays that require a larger number of cells and higher transfection efficiencies. Although intranuclear microinjections require an investment of equipment and time, the ability to achieve high levels of heterologous protein expression in a physiologically relevant environment makes this technique a very useful tool to investigate protein function.

Direct intranuclear injection of cDNA is an effective transfection technique for post-mitotic cells. This method provides high levels of heterologous protein expression from single or multiple cDNA constructs and enables protein function to be studied in a physiologically relevant environment with a variety of single cell assays.