وأيد هذا الدراسة المنح المقدمة من الجمعية الوطنية للتصلب المتعدد (RG4591A1/2)، والمعاهد الوطنية للصحة (R03NS06740402). يشكر المؤلفون الدكتور إيفان هيرنانديز وأفراد المعمل لتوفير مساحة المختبرات والمعدات والمشورة.

الكتاب قد لا الكشف.

في هذه الرسالة، نقدم طريقة لعزل الماوس غير ناضجة جداً المنقي oligodendrocyte الثقافات تتسم بالكفاءة. بالمقارنة مع البروتوكولات المنشورة سابقا39،40، أثمر هذا الأسلوب درجة نقاء أعلى بمستوى أقل بكثير من الإيجابية توصيني astrocytes ونسبة منخفضة جداً من الخلايا الأخرى غير ?...

Oligodendrocytes (عملية شريان الحياة) هي خلايا ميليناتينج الجهاز العصبي المركزي (CNS)1. بالعزلة وثقافة oligodendrocytes الأولية في بيئة محكم تنظيم أداة قيمة للدراسة في المختبر لتطوير أوليجوديندروجليا، فضلا عن بيولوجيا دملينتينغ الأمراض مثل التصلب المتعدد2 . وهذا يتطلب oligodendrocyte فعالة وقوية العزلة وثقافة أسلوب3. في هذه الدراسة، ونحن استغل للتعبير عن علامة سطح الخلية oligodendrocyte المميزة لتنفيذ تقنية عزل معدلة التي سريعة ومحددة.
وقد تم التعرف على أربع مراحل متميزة من النضج أوليجوديندروسيتي، بعضها يتسم بالتعبير عن علامات سطح الخلية المميزة لكل مرحلة من مراحل النمو (الشكل 1). هذه العلامات السطحية الخلية يمكن التعرف عليه بواسطة أجسام مضادة محددة4،5، ويمكن استخدامها لعزل عملية شريان الحياة في مراحل محددة. وفي المرحلة الأولى، oligodendrocyte خلايا السلائف (OPCs) لديها القدرة على تتكاثر وتهاجر، وتحديداً التعبير عن عامل النمو المشتقة من الصفيحات مستقبلات (بدجف-Rα)6، جانجليوسيدي A2B5، وبروتيوغليكان NG27،8 ، بوليسياليك حمض العصبية خلية التصاق جزيء9 والأحماض الدهنية-ملزمة البروتين 7 (FABP7)10. وقد المعاونة مورفولوجيا القطبين مع بعض العمليات قصيرة النابعة من أقطاب المعارضة لخلايا الجسم، والتي من سمات الخلايا العصبية السلائف11.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57543/57543fig1v2.jpg"> رقم 1: التعبير عن علامات سطح الخلية أثناء وضع الماوس أوليجوديندروسيتي. عملية شريان الحياة الخلية السطحية علامات مثل A2B5، GalC (O1)، NG2، O4، وبدجف-Rα يمكن استخدامها لعزل oligodendrocytes على وجه التحديد في مرحلة إنمائية محددة باستخدام أجسام مضادة محددة.   <a href="//cloudflare.jove.com/files/ftp_upload/57543/57543fig1v2large.jpg" target="_blank">الرجاء انقر هنا لمشاهدة نسخة أكبر من هذا الرقم-</a>
وفي المرحلة الثانية، تثير بريوليجوديندروسيتيس المعاونة ويعرب في غشاء الخلية علامات حزب مؤتمر اودوا الشعبي، ليس فقط، بل أيضا سولفاتيدي (جالاكتوليبيد مسلفت) المعترف بها من قبل ال12،جسم O413، بروتين GPR1714، التي استمرت حتى المرحلة oligodendrocyte غير ناضجة (الرتب الأخرى). وفي هذه المرحلة، تمديد بريوليجوديندروسيتيس العمليات قصيرة متعدد الأقطاب. بريوليجوديندروسيتيس هي مرحلة را رئيسيا في يوم الولادة 2 (P2) في هذا الشأن الأبيض الدماغي الجرذ والفأر مع سكان صغيرة غير ناضجة عملية شريان الحياة15.
خلال المرحلة الثالثة، وتواصل غير ناضجة عملية شريان الحياة التعبير عن O4 وتفقد التعبير عن علامات A2B5 وفي NG2 ويبدأ بالتعبير عن جالاكتوسيريبروسيدي ج16. في هذه المرحلة، ملتزمون بنسب أوليجوديندروجليال عملية شريان الحياة وتصبح الخلايا الانقسامية اللاحقة مع فروع متشعب طويل17،18. را غير ناضجة تشكل أكثر من 80 في المائة من هذه المسألة الأبيض القوارض في P7 وتحترم الخلايا MBP+ الأولى في هذا الوقت15،19،،من2021. ولذلك، يمكن ضمان عزل عملية شريان الحياة في P7 عالية الغلة الخلوية.
في المرحلة الرابعة والنهائية من التنمية را، أعرب شريان ناضج ميليناتينج البروتينات (البروتين الأساسية المايلين (MBP) والبروتين بروتيوليبيد (حزب العمال التقدمي) وبروتين سكري المايلين المرتبطة (ماج) والمايلين أوليجوديندروسيتي بروتين سكري (موج)22،23 ،24،،من2526. في هذه المرحلة، تمديد هذا الاتفاق شكل انورابينج مانعات حول محاور عصبية الأغشية شريان ناضج وقادرون على ميلينت. ويتزامن هذا مع ملاحظة أن الخلايا MBP+ في الدماغ الفئران والفأر، تصبح وفرة متزايدة في P1419،،من2021.
منذ عزل أول oligodendrocyte فوستر والزملاء في عام 196727، نفذت عدة طرق لعزل عملية شريان الحياة من القوارض الجهاز العصبي المركزي بما في ذلك إيمونوبانينج28،،من2930، الأسفار تنشيط الخلية الفرز (نظام مراقبة الأصول الميدانية) استغلال الخلية الخاصة بسطح المستضدات28،31، الطرد المركزي التدرج التفاضلية32،،من3334،35 وأسلوب هز على أساس الانضمام التفاضلية لإطلاق الجهاز العصبي المركزي مختلفة36،37. ومع ذلك، أساليب الثقافة القائمة قد القيود، لا سيما من حيث النقاء والغلة والوقت المطلوب لتنفيذ إجراءات38. ولذلك، أكثر كفاءة أساليب معزل oligodendrocytes المطلوبة.
في هذه الورقة، نقدم بسيطة وتحقيق الكفاءة في اختيار الأسلوب لعزل إيمونوماجنيتيك المرحلة O4 ثلاث+ الخلايا بريوليجوديندروسيتيس من الجراء الفئران حديثي الولادة. هذا الأسلوب تعديل التقنيات التي ذكرت باميرى et al. 39 ودينكمان et al. 40 ويوفر نقاء إعداد oligodendrocyte أكثر من 80 في المائة في حوالي 4 ح.

<table>
	<tbody>
		<tr>
			<td>10ml serological pipets</td>
			<td>Fisher Scientific</td>
			<td>13-676-10J</td>
			<td></td>
		</tr>
		<tr>
			<td>10ml syringe Luer-Loc tip</td>
			<td>BD, Becton Dickinson</td>
			<td>309604</td>
			<td></td>
		</tr>
		<tr>
			<td>15ml conical tubes</td>
			<td>Falcon</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well tissue culture plates</td>
			<td>Falcon</td>
			<td>353935</td>
			<td></td>
		</tr>
		<tr>
			<td>40&#181;m cell strainer</td>
			<td>Fisher Scientific</td>
			<td>22368547</td>
			<td></td>
		</tr>
		<tr>
			<td>50ml conical tubes</td>
			<td>Falcon</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>5ml serological pipets</td>
			<td>Fisher Scientific</td>
			<td>13-676-10H</td>
			<td></td>
		</tr>
		<tr>
			<td>60mm tissue culture plates</td>
			<td>Falcon</td>
			<td>353002</td>
			<td></td>
		</tr>
		<tr>
			<td>70&#181;m cell strainer</td>
			<td>Fisher Scientific</td>
			<td>22363548</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 goat anti-mouse IgG (H+L) secondary antibody</td>
			<td>Invitrogen</td>
			<td>A11001</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 goat anti-rabbit IgM (H+L) secondary antibody</td>
			<td>Invitrogen</td>
			<td>A21042</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 goat anti-rabbit IgM (H+L) secondary antibody</td>
			<td>Invitrogen</td>
			<td>A11008</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 goat anti-chicken IgG (H+L) secondary antibody</td>
			<td>Invitrogen</td>
			<td>A11042</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-O4 beads- Anti-O4MicroBeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-094-543</td>
			<td></td>
		</tr>
		<tr>
			<td>Apo-Transferrin human</td>
			<td>Sigma</td>
			<td>T1147</td>
			<td></td>
		</tr>
		<tr>
			<td>Autofil complete bottle top filter assembly, 0.22um filter, 250ml</td>
			<td>USA Scientific</td>
			<td>6032-1101</td>
			<td></td>
		</tr>
		<tr>
			<td>Autofil complete bottle top filter assembly, 0.22um filter, 250ml</td>
			<td>USA Scientific</td>
			<td>6032-1102</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 Supplement</td>
			<td>Invitrogen</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>Sigma</td>
			<td>B7660</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Growth Serum (BGS)</td>
			<td>GE Healthcare Life Sciences</td>
			<td>SH30541.03</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Fisher Scientific</td>
			<td>BP-1600-100</td>
			<td></td>
		</tr>
		<tr>
			<td>CNTF</td>
			<td>Peprotech</td>
			<td>450-50</td>
			<td></td>
		</tr>
		<tr>
			<td>d-Biotin</td>
			<td>Sigma</td>
			<td>B4639</td>
			<td></td>
		</tr>
		<tr>
			<td>Desoxyribonuclease I (DNAse I)</td>
			<td>Worthington</td>
			<td>LS002007</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Fisher Scientific</td>
			<td>S311</td>
			<td></td>
		</tr>
		<tr>
			<td>Epifluorescence microscope with an Olympus DP70 camera</td>
			<td>Olympus</td>
			<td>Bx51</td>
			<td></td>
		</tr>
		<tr>
			<td>Feather disposable scalpels</td>
			<td>Andwin Scientific</td>
			<td>EF7281C</td>
			<td></td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>Sigma</td>
			<td>F6886</td>
			<td></td>
		</tr>
		<tr>
			<td>German glass coverslips, #1 thickness, 12mm diameter round</td>
			<td>NeuVitro</td>
			<td>GG-12-oz</td>
			<td></td>
		</tr>
		<tr>
			<td>GFAP antibody</td>
			<td>Aves</td>
			<td>GFAP</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Fisher Scientific</td>
			<td>D16-1</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Invitrogen</td>
			<td>35050-61</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Invitrogen</td>
			<td>12585-014</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic separator stand - MACS multistand</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic separator-MiniMACS separator</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-302</td>
			<td></td>
		</tr>
		<tr>
			<td>Millex PES 0.22&#181;m filter unit</td>
			<td>Millipore</td>
			<td>SLG033RS</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting media- Prolong Gold with DAPI</td>
			<td>Thermo Fisher</td>
			<td>P36930</td>
			<td></td>
		</tr>
		<tr>
			<td>N-acetyl-cysteine (NAC)</td>
			<td>Sigma</td>
			<td>A8199</td>
			<td></td>
		</tr>
		<tr>
			<td>Natural mouse laminin</td>
			<td>Invitrogen</td>
			<td>23017-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal Medium A</td>
			<td>Invitrogen</td>
			<td>10888-022</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurotrophin-3 (NT-3)</td>
			<td>Peprotech</td>
			<td>450-03</td>
			<td></td>
		</tr>
		<tr>
			<td>NG2 antibody</td>
			<td>Millipore</td>
			<td>AB5320</td>
			<td></td>
		</tr>
		<tr>
			<td>Papain</td>
			<td>Worthington</td>
			<td>LS003126</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS without Ca2+ and Mg2+</td>
			<td>Sigma</td>
			<td>D5652</td>
			<td></td>
		</tr>
		<tr>
			<td>PDGF</td>
			<td>Peprotech</td>
			<td>100-13A</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Falcon</td>
			<td>351029</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-Lysine</td>
			<td>Sigma</td>
			<td>P6407</td>
			<td></td>
		</tr>
		<tr>
			<td>Primocin</td>
			<td>Invivogen</td>
			<td>ant-pm-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma</td>
			<td>P8783</td>
			<td></td>
		</tr>
		<tr>
			<td>Putrescine</td>
			<td>Sigma</td>
			<td>P5780</td>
			<td></td>
		</tr>
		<tr>
			<td>Selection column-LS columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Selenite</td>
			<td>Sigma</td>
			<td>S5261</td>
			<td></td>
		</tr>
		<tr>
			<td>Trace elements B</td>
			<td>Corning</td>
			<td>25-000-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Triiodothyronine (T3)</td>
			<td>Sigma</td>
			<td>T6397</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Solution</td>
			<td>Corning</td>
			<td>25-900-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr>
			<td>B27NBMA</td>
			<td></td>
			<td></td>
			<td>487.75 mL Neurobasal Medium A; 10 mL B27 Supplement; 1 mL Primocin; 1.25 mL Glutamax; Filter sterilize and store at 4 &#176;C until use.</td>
		</tr>
		<tr>
			<td>B27NBMA + 10% BGS</td>
			<td></td>
			<td></td>
			<td>27 mL B27NBMA; 3 mL Bovine growth serum</td>
		</tr>
		<tr>
			<td>CNTF solution stock (10 &#181;g/ml; 1000X)</td>
			<td></td>
			<td></td>
			<td>Order from Peprotech (450-50). Make up at 0.1 to 1 mg/ml according to Manufacturer&#8217;s instruction (may vary from lot to lot) in buffer (e.g. DPBS + 0.2% BSA). Store at -80 &#176;C. 
			Working solution (10 &#181;g/ml, 1000X) 
			1. Make on 0.2% BSA (Fisher scientific BP-1600-100) in DPBS solution and filter sterilize. 
			2. Dilute master stock aliquot to 10&#181;g/ml in sterile, chilled 0.2% BSA/DPBS. 
			3. Aliquot (20&#181;l/tube) and snap freeze in liquid nitrogen. 
			4. Store aliquots at -80 &#176;C.</td>
		</tr>
		<tr>
			<td>d-Biotin stock solution (50 &#181;g/ml; 5000X)</td>
			<td></td>
			<td></td>
			<td>Resuspend d-Biotin (Sigma-B4639) in double-distilled H2O at 50 &#181;g/ml (e.g. 2.5 mg in 50 ml of ddH2O). Resuspension might take fair amount of agitation/vortexing, or mild warming briefly at 37&#176;C. If the d-Biotin still will not solubilize, it is fine to make up a less concentrated (e.g. 10&#181;g/ml), and to add a higher volume to the B27NBMA (1/1000), instead of 1/5000). Store at 4&#176;C.</td>
		</tr>
		<tr>
			<td>DNase I stock solution</td>
			<td></td>
			<td></td>
			<td>1. Dissolve at 12,500 U Deoxyribonuclease I / ml in HBSS chilled on ice. 
			2. Filter sterilize on ice 
			3. Aliquot at 200 &#181;l and freeze overnight at -20&#176;C. 
			4. Store aliquots at -20 to -30&#176;C.</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline (w/o Ca2+ and Mg2+)</td>
			<td></td>
			<td></td>
			<td>Dissolve pouch in 1 Liter of water to yield 1 liter of medium at 9.6 grams of powder per liter of medium. Store at 2-8 &#176;C.</td>
		</tr>
		<tr>
			<td>Forskolin stock solution (4.2 mg/ml; 1000X)</td>
			<td></td>
			<td></td>
			<td>Add 1 ml of sterile DMSO to 50 mg Forskolin in bottle (Sigma-F6886) and pipette until resuspended. Transfer to a 15 ml centrifuge tube and add 11 ml of sterile DMSO to bring to 4.2 mg/ml. Aliquot (e.g. 20 &#181;l) and store at -20&#176;C.</td>
		</tr>
		<tr>
			<td>Hank&#8217;s balanced salts (HBSS) (Sigma</td>
			<td></td>
			<td></td>
			<td>1. Measure 900 ml of water (temperature 15-20 &#176;C) in a cylinder and stir gently. 
			2. Add the power and stir until dissolved. 
			3. Rinse original package with a small amount of water to remove all traces of the powder. 
			4. Add to the solution in step 2. 
			5. Add 0.35 gr of sodium bicarbonate (7.5% w/v) for each liter of final volume. 
			6. Keep stirring until dissolved. 
			7. Adjust the pH of the buffer while stirring to 0.1-0.3 units below pH= 7.4 since it may rise during filtration. The use of 1N HCl or 1N NaOH is recommended to adjust the pH. 
			8. Add additional water to bring the final volume to 1L. 
			9. Sterilize by filtration using a membrane with a porosity of 0.22 microns. 
			10. Store at 2-8 &#176;C.</td>
		</tr>
		<tr>
			<td>Insulin stock solution (4000 &#181;g/ml)</td>
			<td></td>
			<td></td>
			<td>Thaw the bottle and aliquot 25 &#181;l per microcentrifuge tube and store at -20&#176;C.</td>
		</tr>
		<tr>
			<td>Laminin solution</td>
			<td></td>
			<td></td>
			<td>Slowly thaw laminin in the cold (2&#176;C to 8&#176;C) to avoid gel formation. Then, aliquot into polypropylene tubes. Store at 5&#176; C to -20&#176; C in aliquots (e.g. 20 &#181;l) and do not freeze/thaw repeatedly. Laminin may be stored at these temperatures for up to six months.</td>
		</tr>
		<tr>
			<td>Magnetic Cell Sorting (MCS) Buffer</td>
			<td></td>
			<td></td>
			<td>Prepare the solution containing phosphate-buffered saline (PBS), pH 7.2, and 0.5% bovine serum albumin (BSA), 0.5 mM EDTA, 5&#181;g/ml Insulin, 1 g/L Glucose. Sterilize and degas by filtration the buffer by passing it through a 0.22 &#181;m Millex filter. Store the buffer at 4&#176;C until use</td>
		</tr>
		<tr>
			<td>N-Acetyl-L-cysteine (NAC) stock solution (5mg/ml; 1000X)</td>
			<td></td>
			<td></td>
			<td>Dissolve N-Acetyl-L-cysteine (Sigma-A8199) at 5 mg/ml in DMEM (e.g. 50 mg NAC in 10 ml B27NBMA). Filter sterilize and aliquot (e.g. 20 &#181;l). Store at -20&#176;C.</td>
		</tr>
		<tr>
			<td>NT3 stock solution (1 &#181;g/ml; 1000X)</td>
			<td></td>
			<td></td>
			<td>Master stock: 
			Order from Peprotech (450-03). Make up at 0.1 to 1 mg/ml according to manufacturer&#8217;s instructions (may vary from lot to lot), in buffer (e.g. DPBS + 0.2% BSA). Store at -80&#176;C. 
			 
			Working stock (1&#181;g/ml; 1000X): 
			1. Make on 0.2% BSA in DPBS solution and filter sterilize. 
			2. Dilute master stock aliquot to 1 &#181;g/ml in sterile, chilled 0.2% BSA/DPBS. 
			3. Aliquot (e.g. 20&#181;l/tube) and snap freeze in liquid nitrogen. 
			4. Store aliquots at -80&#176;C.</td>
		</tr>
		<tr>
			<td>PDGF stock solution (10 &#181;g/ml; 1000X)</td>
			<td></td>
			<td></td>
			<td>Master stock: 
			Order from Peprotech (100-13A). Make up at 0.1 to 1 mg/ml according to manufacturer&#8217;s instructions (may vary from lot to lot) in buffer (e.g. DPBS) + 0.2% BSA). Store at -80&#176;C. 
			 
			Working stock (1&#181;g/ml; 1000X): 
			1. Make on 0.2% BSA in DPBS solution and filter sterilize. 
			2. Dilute master stock aliquot to 1&#181;g/ml in sterile, chilled 0.2% BSA/DPBS. 
			3. Aliquot (e.g. 20&#181;l/tube) and snap freeze in liquid nitrogen. 
			4. Store aliquots at -80&#176;C.</td>
		</tr>
		<tr>
			<td>Poly-D-lysine (1mg/ml; 100X)</td>
			<td></td>
			<td></td>
			<td>Resuspend poly-D-lysine, molecular weight 70-150 kD (Sigma P6407) at 0.5mg/ml in 0.15M boric acid pH 8.4 (e.g. 50mg in 50ml borate buffer). Filter sterilize and aliquot (e.g. 100&#181;l/tube). Store at -20&#176;C. Prior to use, dilute the 100X stock (1mg/ml) to 50 &#181;g/ml in sterile water.</td>
		</tr>
		<tr>
			<td>Oligodendrocyte proliferation media</td>
			<td></td>
			<td></td>
			<td>see Supplementary Table 1</td>
		</tr>
		<tr>
			<td>Oligodendrocyte differentiation media</td>
			<td></td>
			<td></td>
			<td>see Supplementary Table 1</td>
		</tr>
		<tr>
			<td>Sato supplement (100X)</td>
			<td></td>
			<td></td>
			<td>see Supplementary Table 1</td>
		</tr>
		<tr>
			<td>References: the list of reagents and recipes were adopted from the protocols previously described by Emery et. al. 2013 (Emery, B. &#38; Dugas, J. C. Purification of oligodendrocyte lineage cells from mouse cortices by immunopanning. Cold Spring Harb Protoc. 2013 (9), 854-868, doi:10.1101/pdb.prot073973, (2013)) and Dincman et. al. (Dincman, T. A., Beare, J. E., Ohri, S. S. &#38; Whittemore, S. R. Isolation of cortical mouse oligodendrocyte precursor cells. J Neurosci Methods. 209 (1), 219-226, doi:10.1016/j.jneumeth.2012.06.017, (2012))</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

rapid and specific immunomagnetic isolation of mouse primary oligodendrocytes

عزلة فعالة وقوية وثقافة oligodendrocytes الأولية (عملية شريان الحياة) أداة قيمة للدراسة في المختبر لتطوير أوليجوديندروجليا، فضلا عن بيولوجيا دملينتينغ الأمراض مثل التصلب المتعدد و بليزايوس-ميرتسباخر-مثل مرض (PMLD). هنا، فإننا نقدم بسيطة وتحقيق الكفاءة في اختيار الأسلوب لعزل إيمونوماجنيتيك المرحلة O4 ثلاث+ الخلايا بريوليجوديندروسيتيس من الجراء الفئران حديثي الولادة. منذ را غير ناضجة تشكل أكثر من 80% من هذه المسألة الأبيض القوارض في الدماغ عند الولادة يوم 7 (P7) لا يضمن هذا الأسلوب عزل عالية الغلة الخلوية، ولكن أيضا عزلة محددة من شريان الحياة للسودان ملتزمة بالفعل النسب أوليجوديندروجليال، تناقص إمكانية عزل الخلايا تلويث مثل أستروسيتيس والخلايا الأخرى من الدماغ الماوس. هذا الأسلوب هو تعديل التقنيات التي ذكرت سابقا، ويوفر oligodendrocyte إعداد نقاوة أعلى من 80% في حوالي 4 ح.

وكان الغرض من هذه الدراسة لإنشاء أسلوب عزل محسنة ل O4+ أوليجوديندروسيتيس الماوس الأساسية التي تتطلب أقل قدر ممكن التلاعب بالخلايا الهدف. يأخذ الإجراء بأكمله من القتل الرحيم الجراء لطلاء الخلايا في كوفيرسليبس حوالي 4 ح والبيانات الموضحة هنا تمثل ثلاث تجارب مستقلة. ب?...

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Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes

يصف لنا عزلة إيمونوماجنيتيك oligodendrocytes الماوس الأساسي، الذي يسمح لعزل الخلايا في المختبر الثقافة سريعة ومحددة.

عزل إيمونوماجنيتيك سريعة ومحددة من الماوس Oligodendrocytes الأولية

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of ...

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Research

JoVE Journal

Neuroscience

13.5K Views.  State University of New York Downstate Medical Center. يصف لنا عزلة إيمونوماجنيتيك oligodendrocytes الماوس الأساسي، الذي يسمح لعزل الخلايا في المختبر الثقافة سريعة ومحددة.

Video: Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes

Isolation and Culture of Mouse Cortical Astrocytes

Astrocytes are an abundant cell type in the mammalian brain, yet much remains to be learned about their molecular and functional characteristics. In vitro astrocyte cell culture systems can be used to study the biological functions of these glial cells in detail. This video protocol shows how to obtain pure astrocytes by isolation and culture of mixed cortical cells of mouse pups. The method is based on the absence of viable neurons and the separation of astrocytes, oligodendrocytes and microglia, the three main glial cell populations of the central nervous system, in culture. Representative images during the first days of culture demonstrate the presence of a mixed cell population and indicate the timepoint, when astrocytes become confluent and should be separated from microglia and oligodendrocytes. Moreover, we demonstrate purity and astrocytic morphology of cultured astrocytes using immunocytochemical stainings for well established and newly described astrocyte markers. This culture system can be easily used to obtain pure mouse astrocytes and astrocyte-conditioned medium for studying various aspects of astrocyte biology.

Astrocytes have been recognized to be versatile cells participating in fundamental biological processes that are essential for normal brain development and function, and central nervous system repair. Here we present a rapid procedure to obtain pure mouse astrocyte cultures to study the biology of this major class of central nervous system cells.

العزلة وثقافة الخلايا النجمية الماوس القشرية

Astrocytes  are an abundant cell type in the mammalian brain, yet much remains to be  learned about their molecular and functional characteristics. In ...

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

الثقافة الابتدائية من الخلايا العصبية الدوبامين ماوس

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis of primary cultures of neurons. We describe a set of procedures for: labeling newborn mice to be genotyped, rapid genotyping, and establishing low-density cultures of brain neurons from these mice. Individual mice are labeled by tattooing, which allows for long-term identification lasting into adulthood. Genotyping by the described protocol is fast and efficient, and allows for automated extraction of nucleic acid with good reliability. This is useful under circumstances where sufficient time for conventional genotyping is not available, e.g., in mice that suffer from neonatal lethality. Primary neuronal cultures are generated at low density, which enables imaging experiments at high spatial resolution. This culture method requires the preparation of glial feeder layers prior to neuronal plating. The protocol is applied in its entirety to a mouse model of the movement disorder DYT1 dystonia (&#916;E-torsinA knock-in mice), and neuronal cultures are prepared from the hippocampus, cerebral cortex and striatum of these mice. This protocol can be applied to mice with other genetic mutations, as well as to animals of other species. Furthermore, individual components of the protocol can be used for isolated sub-projects. Thus this protocol will have wide applications, not only in neuroscience but also in other fields of biological and medical sciences.

We describe procedures for labeling and genotyping newborn mice and generating primary neuronal cultures from them. The genotyping is rapid, efficient and reliable, and allows for automated nucleic-acid extraction. This is especially useful for neonatally lethal mice and their cultures that require prior completion of genotyping.

رابيد التنميط الجيني للحيوانات تلاه إنشاء الثقافات الأولية من الخلايا العصبية في الدماغ

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis ...

Isolation of Primary Murine Brain Microvascular Endothelial Cells

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional complex of tight and adherens junctions. Together with other cell-types such as astrocytes or pericytes, they form the neurovascular unit (NVU), which specifically regulates the interchange of fluids, molecules and cells between the peripheral blood and the CNS. Through this complex and dynamic system BMECs are involved in various processes maintaining the homeostasis of the CNS. A dysfunction of the BBB is observed as an essential step in the pathogenesis of many severe CNS diseases. However, specific and targeted therapies are very limited, as the underlying mechanisms are still far from being understood. 
Animal and in vitro models have been extensively used to gain in-depth understanding of complex physiological and pathophysiological processes. By reduction and simplification it is possible to focus the investigation on the subject of interest and to exclude a variety of confounding factors. However, comparability and transferability are also reduced in model systems, which have to be taken into account for evaluation. The most common animal models are based on mice, among other reasons, mainly due to the constantly increasing possibilities of methodology. In vitro studies of isolated murine BMECs might enable an in-depth analysis of their properties and of the blood-brain-barrier under physiological and pathophysiological conditions. Further insights into the complex mechanisms at the BBB potentially provide the basis for new therapeutic strategies.
This protocol describes a method to isolate primary murine microvascular endothelial cells by a sequence of physical and chemical purification steps. Special considerations for purity and cultivation of MBMECs as well as quality control, potential applications and limitations are discussed.

Brain microvascular endothelial cells (BMEC) are interconnected by specific junctional proteins forming a highly regulated barrier separating blood and the central nervous system (CNS), the so-called blood-brain-barrier (BBB). The isolation of primary murine brain microvascular endothelial cells, as discussed in this protocol, enables detailed in vitro studies of the BBB.

عزل خلايا الدماغ الأولية الفئران الاوعية الدموية الدقيقة غشائي

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional ...

Rapid and Refined CD11b Magnetic Isolation of Primary Microglia with Enhanced Purity and Versatility

Microglia are the primary responders to central nervous system insults; however, much remains unknown about their role in regulating neuroinflammation. Microglia are mesodermal cells that function similarly to macrophages in surveying inflammatory stress. The classical (M1-type) and alternative (M2-type) activations of macrophages have also been extended to microglia in an effort to better understand the underlying interplay these phenotypes have in neuroinflammatory conditions such as Parkinson&#39;s, Alzheimer&#39;s, and Huntington&#39;s Diseases. In vitro experimentation utilizing primary microglia offers rapid and reliable results that may be extended to the in vivo environment. Although this is a clear advantage over in vivo experimentation, isolating microglia while achieving adequate yields of optimal purity has been a challenge. Common methods currently in use either suffer from low recovery, low purity, or both. Herein, we demonstrate a refinement of the column-free CD11b magnetic separation method that achieves a high cell recovery and enhanced purity in half the amount of time. We propose this optimized method as a highly useful model of primary microglial isolation for the purposes of studying neuroinflammation and neurodegeneration.

Here, we present a protocol to isolate microglia from postnatal mouse pups (day 1) for in vitro experimentation. This improvised method of isolation generates both high yield and purity, a significant advantage over alternate methods that allows broad range experimentation for the purposes of elucidating microglial biology.

عزل المغناطيسي السريع والمكرر CD11b من الابتدائية الخلايا الدبقية الصغيرة مع الطهارة تعزيز وطلاقة

Microglia are the primary responders to central nervous system insults; however, much remains unknown about their role in regulating neuroinflammation. ...

Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has revealed microglia to be dynamic, capable of assuming both pro-inflammatory and anti-inflammatory phenotypes. Both M1 (pro-inflammatory) and M2 (pro-reparative) phenotypes play an important role in neuroinflammatory conditions such as perinatal brain injury, and exhibit differing functions in response to certain environmental stimuli. The modulation of microglial activation has been noted to confer neuroprotection thus suggesting microglia may have therapeutic potential in brain injury. However, more research is required to better understand the role of microglia in disease, and this protocol facilitates that. The protocol described below combines a density gradient centrifugation process to reduce cellular debris, with magnetic separation, producing a highly pure sample of primary microglial cells that can be used for in vitro experimentation, without the need for 2-3 weeks culturing. Additionally, the characterization steps yield robust functional data about microglia, aiding studies to better our understanding of the polarization and priming of these cells, which has strong implications in the field of regenerative medicine.

A protocol for the isolation of primary microglia from murine brains is presented. This technique aids in furthering the current understanding of neurological conditions. Density gradient centrifugation and magnetic separation are combined to produce sufficient yield of a highly pure sample. Furthermore, we outline the steps for characterization of microglia.

توصيف والعزلة للماوس Microglia الأولية بكثافة التدرج الطرد المركزي

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has ...

Isolation of Primary Murine Skeletal Muscle Microvascular Endothelial Cells

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal muscles regulating the exchange of fluids and nutrients as well as the immune response against infectious agents by controlling immune cell migration. For these functions, MMEC form a functional &#34;myovascular unit&#34; (MVU), with further cell types, such as fibroblasts, pericytes and skeletal muscle cells. Consequently, a dysfunction of MMEC and therefore the MVU contributes to a vast variety of myopathies. However, regulatory mechanisms of MMEC in health and disease remain insufficiently understood and their elucidation precedes more specific treatments for myopathies. The isolation and in-depth investigation of primary MMEC functions in the context of the MVU might facilitate a better understanding of these processes.
This article provides a protocol to isolate primary murine MMEC of the skeletal muscle by mechanical and enzymatic dissociation including purification and culture maintenance steps.

Microvascular endothelial cells of skeletal muscles (MMEC) shape the inner wall of muscle capillaries and regulate both, exchange of fluids/molecules and migration of (immune) cells between muscle tissue and blood. Isolation of primary murine MMEC, as described here, enables comprehensive in vitro investigations of the &#34;myovascular unit&#34;.

عزل خلايا بطانية Microvascular الابتدائي مورين الهيكل العظمى والعضلات

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal ...

Rapid Isolation of Dorsal Root Ganglion Macrophages

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after peripheral nerve injury. Peripheral monocytic cells, including macrophages, are known to respond to a tissue injury through phagocytosis, antigen presentation, and cytokine release. Emerging evidence has implicated the contribution of dorsal root ganglia macrophages to neuropathic pain development and axonal repair in the context of nerve injury. Rapidly phenotyping (or &#8220;rapid isolation of&#8221;) the response of dorsal root ganglia macrophages in the context of nerve injury is desired to identify the unknown neuroimmune factors. Here we demonstrate how our lab rapidly and effectively isolates macrophages from the dorsal root ganglia using an enzyme-free mechanical dissociation protocol. The samples are kept on ice throughout to limit cellular stress. This protocol is far less time consuming compared to the standard enzymatic protocol and has been routinely used for our Fluorescence-activated Cell Sorting analysis.

Here we present a mechanical dissociation protocol to rapidly isolate macrophages from the dorsal root ganglion for phenotyping and functional analysis.

العزلة السريعة للضامة الجذرية الدجورة

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after ...

Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive human diseases. Considerable advances have been made to uncover the molecular signatures of homeostatic microglia as well as alterations of their gene expression in response to environmental stimuli. With the advent and maturation of single-cell genomic methodologies, it is increasingly recognized that heterogenous microglia may underlie the diverse roles they play in different developmental and pathological conditions. Further dissection of such heterogeneity can be achieved through efficient isolation of microglia from a given region of interest, followed by sensitive profiling of individual cells. Here, we provide a detailed protocol for the rapid isolation of microglia from different brain regions in a single adult mouse brain hemisphere. We also demonstrate how to use these sorted microglia for plate-based deep single-cell RNA sequencing. We discuss the adaptability of this method to other scenarios and provide guidelines for improving the system to accommodate large-scale studies.

We provide a protocol for isolation of microglia from different dissected regions of an adult mouse brain hemisphere, followed by semi-automated library preparation for deep single-cell RNA sequencing of full-length transcriptomes. This method will help to elucidate functional heterogeneity of microglia in health and disease.

عزل ميكروليا الخاصة بالمنطقة من واحد الكبار الفار الدماغ نصف الكره لعمق خليه واحده التسلسل RNA

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive ...

Generation of Oligodendrocytes and Oligodendrocyte-Conditioned Medium for Co-Culture Experiments

In the central nervous system, oligodendrocytes are well-known for their role in axon myelination, that accelerates the propagation of action potentials through saltatory conduction. Moreover, an increasing number of reports suggest that oligodendrocytes interact with neurons beyond myelination, notably through the secretion of soluble factors. Here, we present a detailed protocol allowing purification of oligodendroglial lineage cells from glial cell cultures also containing astrocytes and microglial cells. The method relies on overnight shaking at 37 &#176;C, which allows selective detachment of the overlying oligodendroglial cells and microglial cells, and the elimination of microglia by differential adhesion. We then describe the culture of oligodendrocytes and production of oligodendrocyte-conditioned medium (OCM). We also provide the kinetics of OCM treatment or oligodendrocytes addition to purified hippocampal neurons in co-culture experiments, studying oligodendrocyte-neuron interactions.

Herein, we display an efficient method for the purification of oligodendrocytes and production of oligodendrocyte-conditioned medium that can be used for co-culture experiments.

جيل من Oligodendrocytes وOligodendrocyte- متوسطة مكيفة للتجارب الثقافة المشتركة

In the central nervous system, oligodendrocytes are well-known for their role in axon myelination, that accelerates the propagation of action potentials ...