Name: primary microglia isolation from mixed glial cell cultures of neonatal rat brain tissue
Uploaded: 2012-08-15T12:00:00
Description: Watch this Scientific Journal Video about Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue at JoVE.com

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1. Dissection des tissus de cerveau de rat nouveau-né <ol><li> Refroidissement Leibovitz L-15 milieux conditionnés (Leibowitz L-15 + 0,1% de BSA + Pen 1% / Strep) à 4 ° C. Milieux de culture chaudes (DMEM + 10% de FBS + 1% de pénicilline / streptomycine) à 37 ° C. Préparer 4 60x15 mm boîtes de Pétri avec 4-5 ml de L-15 climatisées médias sur la glace. Stériliser tous les instruments chirurgicaux avec de l&#39;éthanol 100%. </li><li> Rincer les chiots nouveau-nés P2 rap avec de l&#39;éthanol 70%. (Raton...

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	<li>Ransohoff, R. M., Perry, V. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+physiology:+unique+stimuli,+specialized+responses.">Microglial physiology: unique stimuli, specialized responses.</a> Annu. Rev. Immunol. 27, 119-145 (2009).</li><li>Raivich, G., Bohatschek, M., Kloss, C. U., Werner, A., Jones, L. L., Kreutzberg, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroglial+activation+repertoire+in+the+injured+brain:+graded+response,+molecular+mechanisms+and+cues+to+physiological+function.">Neuroglial activation repertoire in the injured brain: graded response, molecular mechanisms and cues to physiological function.</a> Brain Res. Brain Res. Rev. 30, 77-105 (1999).</li><li>Loane, D. J., Byrnes, K. 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Alors que ce protocole est régulièrement utilisé pour produire la microglie pur et sain pour les expériences de recherche, un examen attentif des aspects techniques au cours de parties critiques de la procédure permettra de minimiser la variabilité dans la microglie isolée. Tout d&#39;abord, lors de la dissection du tissu cérébral de rats nouveau-nés petits, travaillant en temps opportun est nécessaire pour minimiser les dommages hypoxiques et ischémiques du tissu. Cependant, il est également important d&#3...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Le numéro de catalogue </td><td> Commentaires </td></tr><tr><td> 60 mm x 15 mm boîtes de Pétri </td><td> Fisher marque </td><td> 0875713A </td><td></td></tr><tr><td> De Sharp ciseaux de dissection </td><td> Outils belle science </td><td> 14094-11 </td><td></td></tr><tr><td> Dumont # 7b pince standard des conseils, des courbes, 11cm </td><td> Outils belle science </td><td> 11270-20 </td><td></td></tr><tr><td> Dumont # 5 pince standard des conseils, des droites, 11 cm </td><td> Outils belle science </td><td> 11251-10 </td><td></td></tr><tr><td> Tubes de 50 ml à centrifuger coniques </td><td> VWR </td><td> 89039-656 </td><td></td></tr><tr><td> 5 pipettes sérologiques ml </td><td> Grenier Bio One </td><td> 606180 </td><td></td></tr><tr><td> 10 pipettes sérologiques ml </td><td> Grenier Bio One </td><td> 607180 </td><td></td></tr><tr><td> 100 um crépine stérile cellule de nylon </td><td> Faucon </td><td> 35-2360 </td><td></td></tr><tr><td> 75 cm 2 flacons de culture tissulaire </td><td> Corning </td><td> 430641 </td><td></td></tr><tr><td> Dulbecco milieu essentiel minimal (DMEM) </td><td> Gibco (Invitrogen) </td><td> 31053-028 </td><td></td></tr><tr><td> Leibovitz milieu L-15 </td><td> Gibco (Invitrogen) </td><td> 11415064 </td><td></td></tr><tr><td> Sérum de veau fœtal </td><td> Gibco (Invitrogen) </td><td> 16000-036 </td><td></td></tr><tr><td> Fœtale sérum équin </td><td> Pêcheur </td><td> SH3007402 </td><td></td></tr><tr><td> La pénicilline-streptomycine </td><td> Gibco (Invitrogen) </td><td> 15140163 </td><td></td></tr><tr><td> L&#39;éthanol à 100% </td><td> La Société Warner Graham </td><td> 64-17-5 </td><td></td></tr><tr><td> Phosphate solution saline tamponnée, 10X, pH 7,4 </td><td> Qualité biologique, inc. </td><td> 119-069-131 </td><td> 1X dans l&#39;eau distillée stérile </td></tr><tr><td> Sacs de Biohazard </td><td> VWR </td><td> 14220-028 </td><td></td></tr><tr><td> Hémocytomètre </td><td> Hausser scientifique </td><td> 1492 </td><td></td></tr><tr><td> Des plaques 6 puits culture cellulaire avec une surface CellBIND </td><td> Corning </td><td> 3335 </td><td></td></tr></tbody></table>

primary microglia isolation from mixed glial cell cultures of neonatal rat brain tissue

Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity 1,2. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection 3. Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop 4-6. As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted.
	Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study.
	The principle and protocol of this methodology have been described in the literature 7,8. Additionally, alternate methodologies to isolate primary microglia are well described 9-12. Homogenized brain tissue may be separated by density gradient centrifugation to yield primary microglia 12. However, the centrifugation is of moderate length (45 min) and may cause cellular damage and activation, as well as, cause enriched microglia and other cellular populations. Another protocol has been utilized to isolate primary microglia in a variety of organisms by prolonged (16 hr) shaking while in culture 9-11. After shaking, the media supernatant is centrifuged to isolate microglia. This longer two-step isolation method may also perturb microglial function and activation. We chiefly utilize the following microglia isolation protocol in our laboratory for a number of reasons: (1) primary microglia simulate in vivo biology more faithfully than immortalized rodent microglia cell lines, (2) nominal mechanical disruption minimizes potential cellular dysfunction or activation, and (3) sufficient yield can be obtained without passage of the mixed glial cell cultures.
	It is important to note that this protocol uses brain tissue from neonatal rat pups to isolate microglia and that using older rats to isolate microglia can significantly impact the yield, activation status, and functional properties of isolated microglia. There is evidence that aging is linked with microglia dysfunction, increased neuroinflammation and neurodegenerative pathologies, so previous studies have used ex vivo adult microglia to better understand the role of microglia in neurodegenerative diseases where aging is important parameter. However, ex vivo microglia cannot be kept in culture for prolonged periods of time. Therefore, while this protocol extends the life of primary microglia in culture, it should be noted that the microglia behave differently from adult microglia and in vitro studies should be carefully considered when translated to an in vivo setting.

Watch this Scientific Journal Video about Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue at JoVE.com

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

Isoler les microglies primaire à partir de l&#39;hétérogénéité cellulaire du cerveau est essentielle pour enquêter sur leur rôle dans les deux conditions physiologiques et pathologiques. Ce protocole décrit une isolation mécanique et technique mixte de culture cellulaire qui fournit un rendement élevé et de haute pureté, viables primaires pour les cellules microgliales<em&gt; In vitro</em&gt; Étude et les applications en aval.

Isolement primaire microglies de cultures mixtes de cellules gliales de tissus de cerveau de rat nouveau-né

Microglia account for approximately 12% of the  total cellular population in the mammalian brain. While neurons and astrocytes are considered  the major ...

Research

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