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

بتمويل من برنامج جماعية في جامعة الخدمات النظامية.

1. تشريح من المواليد الجدد الجرذ أنسجة المخ <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> البرد ليبوفيتز لL-15 وسائل الإعلام مكيفة (ليبوفيتز L-15 + 0.1٪ BSA + 1٪ القلم / بكتيريا) إلى 4 درجة مئوية. وسائل الإعلام ثقافة الدافئة (DMEM + 10٪ + 1٪ FBS البنسلين / الستربتومايسين عقار) إلى 37 ?...

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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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في حين يستخدم بصورة روتينية على هذا البروتوكول لإنتاج الخلايا الدبقية الصغيرة نقية وصحية للتجارب البحثية، وإمعان النظر في الجوانب الفنية خلال المراحل الحاسمة من الإجراء الحد من التقلبات في الخلايا الدبقية الصغيرة المعزولة. الأول، أثناء تشريح أنسجة الدماغ من الفئر...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم كاشف </td><td> شركة </td><td> فهرس العدد </td><td> تعليقات </td></tr><tr><td> 60 مم × 15 مم أطباق بتري </td><td> فيشر العلامة التجارية </td><td> 0875713A </td><td></td></tr><tr><td> مقص حاد تشريح </td><td> أدوات العلم غرامة </td><td> 14094-11 </td><td></td></tr><tr><td> دومون # 7B ملقط القياسية نصائح، منحنية، 11cm </td><td> أدوات العلم غرامة </td><td> 11270-20 </td><td></td></tr><tr><td> دومون # 5 ملقط القياسية نصائح، على التوالي، 11 سم </td><td> أدوات العلم غرامة </td><td> 11251-10 </td><td></td></tr><tr><td> 50 مل من أنابيب الطرد المركزي مخروطي </td><td> VWR </td><td> 89039-656 </td><td></td></tr><tr><td> 5 الماصات مل المصلية </td><td> جرينير بيو واحد </td><td> 606180 </td><td></td></tr><tr><td> 10 مل الماصات المصلية </td><td> جرينير بيو واحد </td><td> 607180 </td><td></td></tr><tr><td> 100 ميكرون خلية النايلون معقم مصفاة </td><td> صقر </td><td> 35-2360 </td><td></td></tr><tr><td> 75 سم 2 قوارير زراعة الأنسجة </td><td> كورنينج </td><td> 430641 </td><td></td></tr><tr><td> Dulbecco والمتوسطة الأساسية الدنيا (DMEM) </td><td> Gibco (إينفيتروجن) </td><td> 31053-028 </td><td></td></tr><tr><td> ليبوفيتز لL-15 متوسطة </td><td> Gibco (إينفيتروجن) </td><td> 11415064 </td><td></td></tr><tr><td> مصل بقري جنيني </td><td> Gibco (إينفيتروجن) </td><td> 16000-036 </td><td></td></tr><tr><td> الجنين فرسي المصل </td><td> الصياد </td><td> SH3007402 </td><td></td></tr><tr><td> البنسلين الستربتوميسين </td><td> Gibco (إينفيتروجن) </td><td> 15140163 </td><td></td></tr><tr><td> 100٪ الإيثانول </td><td> شركة وارنر جراهام </td><td> 64-17-5 </td><td></td></tr><tr><td> مخزنة فوسفات حل، 10X المالحة، ودرجة الحموضة 7.4 </td><td> جودة البيولوجي، المؤتمر الوطني العراقي. </td><td> 119-069-131 </td><td> 1X في الماء، ومعقم مقطر </td></tr><tr><td> أكياس واقية </td><td> VWR </td><td> 14220-028 </td><td></td></tr><tr><td> Haemocytometer </td><td> Hausser العلمية </td><td> 1492 </td><td></td></tr><tr><td> 6 كذلك لوحات ثقافة الخلية مع سطح cellBIND </td><td> كورنينج </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

عزل الخلايا الدبقية الصغيرة الأولية من عدم التجانس الخلوية من الدماغ ضروري للتحقيق في دورها في كل الظروف الفسيولوجية والمرضية. هذا البروتوكول يصف العزلة الميكانيكية والمختلطة تقنية زراعة الخلايا التي توفر عائدات عالية ونقاء عالية، وقابلة للحياة خلايا دبقية صغيرة الرئيسي لل<em&gt; في المختبر</em&gt; الدراسة والتطبيقات المصب.

عزل الخلايا الدبقية الصغيرة الابتدائية المختلطة من زراعات الخلايا الغروية من المواليد الجدد الجرذ أنسجة المخ

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

Research

JoVE Journal

Immunology and Infection

Isolation of Brain-infiltrating Leukocytes

عزل الكريات البيضاء في الدماغ التسلل

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine ...

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

وهناك طريقة بسيطة وفعالة لعزل تقوم البلاعم من الثقافات الابتدائية المختلطة من خلايا الكبد الكبار

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

جيل من الثقافات الطوافة عضوي النمط من الخلايا الكيراتينية الإنسان الأساسية

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

الخلايا الدبقية الصغيرة زراعة من الجهاز العصبي المركزي حديثي الولادة والكبار

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

عزل الخلايا الجذعية متعلق بالنخاع الشوكي والخلايا الظهارية من الغدة الصعترية الإنسان

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates

عزل الخلايا الدبقية الصغيرة القشرية مع نمط ظاهري مناعي المحفوظة وظيفة من الفئران حديثي الولادة

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure ...

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

نموذج غير الغازية من ممرض للأعصاب<em&gt; الإشريكية القولونية</em&gt; العدوى في الأطفال حديثي الولادة الجرذ

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

عزل وحقن في الوريد من الفئران نخاع العظام المستمدة حيدات

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Isolation of Sertoli Cells and Peritubular Cells from Rat Testes

عزل خلايا سيرتولي وPeritubular الخلايا من الفئران الخصية

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

العزلة وتحليل تدفق Cytometric من الخلايا المناعية من الدماغ الدماغية ماوس

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...