Name: establishing primary adult fibroblast cultures from rodents
Uploaded: 2010-10-05T17:00:00
Description: Watch this Scientific Journal Video about Establishing Primary Adult Fibroblast Cultures From Rodents Video at JoVE.com

<p class="jove_content">We thank Dr. Steven Austad for providing us with the first version of this protocol.  This work was supported by grants from the NIH and the Ellison Medical Foundation to V.G. and A.S.</p>

<p class="jove_title">1. Before Starting</p>
<ol>
  <li> Sterilize scissors and forceps with 70% ethanol.</li>
  <li> Place small magnetic stirrer inside a 30 mL beaker, cover with two layers of foil, and autoclave.</li>
  <li> Prepare a 28 Wunsch units/mL stock solution of Liberase Blendzyme 3 in sterile water. Make 0.5 mL aliquots and freeze at -20&deg;C. Thaw a new aliquot before every use. The solution may appear cloudy after thawing. Vortex the solution until it becomes clear.</li>
  <li> Warm up the cell culture media.</li>
</ol>
<...

<ol>
	<li>Seluanov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+tumor+suppressor+mechanisms+evolve+in+rodent+species+that+differ+in+size+and+lifespan.+Aging+.">Distinct tumor suppressor mechanisms evolve in rodent species that differ in size and lifespan. Aging .</a> <em style='font-style: italic'>Cell</em>. <b>7</b>, 813-823 (2008).</li><li>Parrinello, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+sensitivity+severely+limits+the+replicative+lifespan+of+murine+fibroblasts.">Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts.</a> <em style='font-style: italic'>Nat Cell Biol</em>. <b>5</b>, 741-747 (2003).</li></ol>

<p class="jove_content">Normal primary fibroblasts provide an excellent alternative to established cancer cell lines in biological research. An important advantage of fibroblasts is that they do not carry mutations in oncogenes and tumor suppressors and maintain intact cell cycle checkpoints. This makes normal fibroblasts a preferred system for the studies of cell cycle regulation, DNA repair, and apoptosis. The protocol described here provides a simple recipe for isolation and maintenance of primary fibroblasts. This protocol was used to succe...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<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>DMEM/F12 media</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11330-032</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum (FBS), Qualified</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>01437-036</td>
<td>The Qualified serum had been pre-tested to provide good growth support for primary fibroblasts.</td>
</tr>
<tr>
<td>Antibiotic/Antimycotic</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15420-096</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td><strong>Liberase TM Research Grade</strong></td>
<td>&nbsp;</td>
<td>Roche</td>
<td><strong>05401127001</strong></td>
<td>Replacement enzyme.</td>
<tr>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>A note from the authors:
Since Roche discontinued Liberase Blendzyme 3 (11814184001), they recommend using Liberase TM Research Grade medium Thermolysin (Cat. no. 05401119001 - 10 mg, Cat. no. 05401127001 - 100mg) instead. We have switched to this enzyme successfully with no issues.</td>
<tr>
<td>EMEM media</td>
<td>&nbsp;</td>
<td>ATCC</td>
<td>30-203</td>
<td>The EMEM media from ATCC already contains nonessential amino acids and sodium pyruvate.</td>
</tr>
<tr>
<td>Feathered #21 disposable, sterile scalpel</td>
<td>&nbsp;</td>
<td>Multiple suppliers</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Three Gas Control incubator</td>
<td>&nbsp;</td>
<td>Forma or Heraeus</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>
	

establishing primary adult fibroblast cultures from rodents

<p class="jove_content">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.</p>

Watch this Scientific Journal Video about Establishing Primary Adult Fibroblast Cultures From Rodents Video at JoVE.com

Establishing Primary Adult Fibroblast Cultures From Rodents Video (Video) | JoVE

<p class="jove_content">This article describes a protocol for isolation and maintenance of primary fibroblast cultures from skin and lung tissue of wild rodents.</p>

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 ...

Research

JoVE Journal

Biology

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae Video (Video) | JoVE

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

Whole Cell Recordings from Brain of Adult Drosophila

Whole Cell Recordings from Brain of Adult Drosophila Video (Video) | JoVE

In this video, we demonstrate the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons. We begin by ...

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates Video (Video) | JoVE

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

Primary Culture of Adult Rat Heart Myocytes

Primary Culture of Adult Rat Heart Myocytes Video (Video) | JoVE

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

Primary Cell Cultures from <em>Drosophila</em> Gastrula Embryos

Primary Cell Cultures from Drosophila Gastrula Embryos Video (Video) | JoVE

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

Streamlined Purification of Plasmid DNA From Prokaryotic Cultures

Streamlined Purification of Plasmid DNA From Prokaryotic Cultures Video (Video) | JoVE

We describe the complete process of AcroPrep Advance Filter Plates for 96 plasmid preparations, starting from prokaryotic culture and ending with high ...

Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures

Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures Video (Video) | JoVE

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system ...

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

NaÃ¯ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures Video (Video) | JoVE

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

Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages

Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages Video (Video) | JoVE

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem ...

Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts

Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts Video (Video) | JoVE

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell ...