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

ממומן על ידי תוכנית עירונית באוניברסיטת שירותי במדים.

1. דיסקציה של רקמת מוח החולדה ילודים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> L-15 צ&#39;יל של ליבוביץ בתקשורת ממוזגים (ליבוביץ L-15 + 0.1% BSA + פן 1% / דלקת) עד 4 ° C. התקשורת והתרבות חמים (DMEM + 10% FBS + 1% פניצילין / סטרפטומיצין) ל 37 ° C. הכן 4 60x15 מ&quot;מ צלחות פטרי עם 4-5 מ&quot;ל של L-15 מד...

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אמנם זה פרוטוקול מנוצל באופן שגרתי כדי לייצר microglia טהור ובריא ניסויים מחקריים, שיקול דעת מעמיק של ההיבטים הטכניים במהלך חלקים קריטיים של תהליך יהיה למזער משונות microglia מבודד. ראשית, במהלך דיסקציה של רקמת המוח מן הגורים חולדה היילודים, עבודה במועד הדרוש כדי למזער את הנז...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם מגיב </td><td> חברה </td><td> מספר קטלוגי </td><td> תגובות </td></tr><tr><td> 60 מ&quot;מ X 15 מ&quot;מ צלחות פטרי </td><td> פישר המותג </td><td> 0875713A </td><td></td></tr><tr><td> מספריים חדים המבתרים </td><td> מדע כלים עדינים </td><td> 14094-11 </td><td></td></tr><tr><td> דומון # מלקחיים סטנדרטיים 7 ב טיפים, מעוגלים, 11cm </td><td> מדע כלים עדינים </td><td> 11270-20 </td><td></td></tr><tr><td> דומונט # 5 מלקחיים סטנדרטיים טיפים, ישרים, 11 ס&quot;מ </td><td> מדע כלים עדינים </td><td> 11251-10 </td><td></td></tr><tr><td> 50 מ&quot;ל צינורות צנטריפוגות חרוטי </td><td> VWR </td><td> 89039-656 </td><td></td></tr><tr><td> 5 מ&quot;ל בטפי להחדרת נוזלים סרולוגיות </td><td> גרנייה ביו אחת </td><td> 606180 </td><td></td></tr><tr><td> 10 מ&quot;ל בטפי להחדרת נוזלים סרולוגיות </td><td> גרנייה ביו אחת </td><td> 607180 </td><td></td></tr><tr><td> תא 100 מיקרומטר סטרילית ניילון מסננת </td><td> בז </td><td> 35-2360 </td><td></td></tr><tr><td> 75 2 ס&quot;מ רקמות תרבות צלוחיות </td><td> קורנינג </td><td> 430641 </td><td></td></tr><tr><td> Dulbecco מינימלית של אמצעי חיוני (DMEM) </td><td> Gibco (Invitrogen) </td><td> 31053-028 </td><td></td></tr><tr><td> ליבוביץ של L-15 בינוני </td><td> Gibco (Invitrogen) </td><td> 11415064 </td><td></td></tr><tr><td> בסרום שור עוברית </td><td> Gibco (Invitrogen) </td><td> 16000-036 </td><td></td></tr><tr><td> עוברי סוסים סרום </td><td> דיג </td><td> SH3007402 </td><td></td></tr><tr><td> פניצילין, סטרפטומיצין </td><td> Gibco (Invitrogen) </td><td> 15140163 </td><td></td></tr><tr><td> 100% אתנול </td><td> חברת וורנר גרהם </td><td> 64-17-5 </td><td></td></tr><tr><td> פוספט שנאגרו תמיסת מלח, 10X, pH 7.4 </td><td> איכות ביולוגית, Inc. </td><td> 119-069-131 </td><td> 1X במים מזוקקים סטרילית </td></tr><tr><td> Biohazard שקיות </td><td> VWR </td><td> 14220-028 </td><td></td></tr><tr><td> Haemocytometer </td><td> האוסר מדעי </td><td> 1492 </td><td></td></tr><tr><td> 6 גם תרבית תאים צלחות עם משטח cellBIND </td><td> קורנינג </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

בידוד microglia העיקרית מן ההטרוגניות הסלולר של המוח חיוני לחקור את תפקידם של שני תנאים פיסיולוגיים ופתולוגיים. פרוטוקול זה מתאר בידוד מכני תאים משולבים בתרבות הטכניקה המספק תשואה גבוהה טוהר גבוהה, תאים קיימא microglial העיקריות<em&gt; במבחנה</em&gt; מחקר ויישומים במורד הזרם.

בידוד ראשי microglia מ מעורבים בתרביות תאים גליה של רקמת מוח החולדה ילודים

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

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