Name: isolation and culture of rodent microglia to promote a dynamic ramified morphology in serum-free medium
Uploaded: 2018-03-09T14:00:00.000+00:00
Duration: 12 min
Description: Watch this Scientific Journal Video about Isolation and Culture of Rodent Microglia to Promote a Dynamic Ramified Morphology in Serum-free Medium at JoVE.com

这项工作得到了克里斯托弗和丹娜基金会国际脊髓损伤研究联合会的支持, dr 和谢尔顿 g. 阿德尔森医学研究基金会, JPB 基金会, 诺华基础研究所,文森特和斯特拉的慷慨贡献, 和达蒙罗扬癌症研究基金会 (DRG-2125-12)。

所有涉及啮齿类动物的程序都符合斯坦福大学的指导方针, 遵守国家和州的法律和政策。所有动物程序都是由斯坦福大学的实验室动物护理管理小组批准的。
注意: 所有解决方案和缓冲区组合都在材料表中提供。
1. 为 CD11b Immunopanning 准备一个培养皿 (0 天)
注: 为每 1-2 只幼鼠大脑准备 1 immunopanning 菜。
<ol><li>在15厘米培养皿中加入25毫升50毫米的三 pH 9.5 溶液。</li>
	<li>添加山羊抗鼠 IgG (H + L 链) 到菜的最后浓度为6µg/毫升。涡流板均匀分布。</li>
	<li>在37摄氏度孵化 1-3 小时的菜肴。</li>
	<li>用带有 Ca2 +和 Mg2 +) 的 DPBS++ (磷酸盐缓冲盐水 (PBS)) 冲洗盘子三次, 然后用包含1µg/mL OX42 抗体的平移缓冲解决方案替换。把盘子在室温下放在平坦的表面上过夜。</li>
</ol>2. 组织收集 (1 天)
注: 该协议应采取〜 3-4 小时。
<ol><li>在开始之前, 确保所有的解决方案是无菌和冰冷却。冷却所有仪器在冰和消毒用乙醇在使用之前。</li>
	<li>遵循适当的监管程序, 用二氧化碳窒息的方式牺牲一只少年实验鼠。 注: 或者, 年轻的动物可能会牺牲的致命剂量氯胺酮/甲苯噻嗪 (100-200 µL 24 毫克/毫升氯胺酮, 2.4 毫克/毫升甲苯噻嗪)。如果动物不到14天的年龄, 必须使用氯胺酮/甲苯噻嗪。如果使用多种动物, 从一种动物中提取组织, 并将其放到冰前的 DPBS++上, 然后再进入随后的动物。</li>
	<li>捏一 hindpaw, 并确保完整的对停止响应从动物之前继续。</li>
	<li>Transcardially 灌注的动物与 10-30 毫升冰冷灌注缓冲使用 27½-G 针, 直到缓冲区运行清楚。 注: 灌注缓冲量根据动物的大小/年龄而定。</li>
	<li>灌注后立即取出动物的头。从脊髓中切开每一侧的枕髁用解剖剪刀, 小心不要损伤大脑。仔细切开每一侧后, 沿顶叶和前额骨切开一侧, 朝向鼻骨。用镊子仔细拉回头骨的顶端, 迅速删除大脑 (或中枢神经系统结构的兴趣), 并放置在冰上的预冷 DPBS++ 。</li>
	<li>对所有剩余的动物重复。 注: 在适当的无菌条件下, 所有步骤都应在层流罩内进行。</li>
</ol>3. 机械离解 (1 天)
<ol><li>在所有的大脑被收集, 把一个大脑切成1毫米3块, 在一个在冰上的培养皿与冷手术刀刀片, 并转移到冰冷的 dounce 均质机与 5-7 毫升冰冷 douncing 缓冲。一次分离一个大脑。</li>
	<li>使用 10-20 的温和和不完全的中风与松散拟合的 dounce 均质体分离组织。注意不要直接粉碎了均质机底部的组织, 而是通过活塞和均质机两侧的空间来推动组织。</li>
	<li>小心地卸下活塞, 防止气泡的引入。允许分离不良的组织块沉淀到均质体的底部, 并将上清转移到新的冷50毫升锥形管上。</li>
	<li>用新鲜的 douncing 缓冲区替换已删除的卷, 并重复步骤3.2 和 3.3, 总共 3-4 回合, 或直到所有组织都已分离。对每个大脑重复这个过程。</li>
</ol>4. 髓鞘摘除 (1 天)
注意: 髓鞘去除是用来隔离小胶质细胞从年龄比 P12 的动物。
<ol><li>用25毫升吸管测量50毫升圆锥管中细胞悬浮液的体积, 然后加入冰冷 douncing 缓冲器, 将总容积调整到33.5 毫升。</li>
	<li>在细胞悬浮液中加入10毫升髓鞘分离缓冲液 (MSB), 并通过多次反转管彻底混合。这将导致23% 最终浓度的 MSB 在43.5 毫升的体积。</li>
	<li>离心细胞为15分钟在 500 x g 在4°c 与缓慢的刹车。 注: 离心机应采取大约 1.5-2 分钟减速。这将产生一个上层的髓鞘和死细胞碎片, 有点阴暗的上清, 和一个小颗粒, 丰富的活细胞。</li>
	<li>用吸管去除髓鞘/碎片和上清上层。卸下顶层时要小心, 以确保尽可能多地删除它。</li>
	<li>并用重悬12毫升的平移缓冲器中的细胞颗粒。轻轻地 triturate 细胞悬浮, 以打破任何可能留下的细胞团簇。</li>
</ol>5. Immunopanning (1 天)
<ol><li>通过70µm 细胞过滤器通过细胞悬浮物来去除大的碎片或细胞团簇。</li>
	<li>用 DPBS++冲洗 OX42-coated 平移盘三次。不要让盘子在洗涤之间完全干燥。</li>
	<li>倒掉最后一个 DPBS++清洗并将过滤后的单元格悬挂应用于平移盘。轻轻地旋转盘子以分配细胞, 然后在室温下在平坦的表面上孵化出20分钟的平板以允许细胞黏附。不要孵育超过20分钟或细胞将变得很难从菜中恢复。</li>
	<li>用 DPBS++ 10 次冲洗平移盘以移除非粘附单元格。小胶质细胞将牢固地附着在盘子上, 所以每冲洗一遍盘子, 以确保去除其他非粘附单元。</li>
	<li>将最后一个 DPBS++清洗并替换为15毫升 DPBS++和 200 ul 胰蛋白酶 (1.25% 个库存解决方案)。</li>
	<li>孵化的菜为≤10分钟在37°c 与 10% CO2胰蛋白酶处理。 注: 不要持续长于10分钟或小胶质细胞将变得难以去除。</li>
	<li>trypsinization 10 分钟后, 小胶质细胞仍会粘在盘子里。倒入胰蛋白酶/DPBS++ , 用 DPBS++轻轻地冲洗 2x, 以除去胰蛋白酶, 代之以12毫升的冰冷小胶质细胞生长培养基 (米高梅)。</li>
	<li>将平移盘放在冰上2分钟, 以帮助削弱细胞/基质的相互作用, 并确保盘子是平的, 以防止平移盘的区域干燥。</li>
	<li>用10毫升的吸管和吸管控制器在高速上强力吸管, 从平移盘中恢复细胞。绘制一个 16 x 16 网格与流的液体从吸管, 试图删除所有的细胞。</li>
	<li>检查显微镜下的细胞在20X 倍放大, 以确保细胞已脱离板块。在盘子的顶端标记斑点, 细胞仍然卡住, 在这些区域重复吹打。</li>
	<li>收集细胞悬浮液和整除 3-4 毫升上清每15毫升圆锥管。旋转15分钟在 500 x g 在4°c 与缓慢的刹车。 注: 小胶质细胞通过少量的体积, 可以最大限度地恢复电池。</li>
	<li>吸入上清液, 将0.5 毫升米高梅与细胞颗粒一起留下。</li>
	<li>并用重悬在剩下的米高梅的每个颗粒, 并汇集所有的细胞从所有的管。</li>
	<li>用 hemocytometer 计数单元格。</li>
	<li>根据应用程序的步骤 6-7 中所述的板单元格。</li>
	<li>文化细胞在37°c 与 10% CO2。 注意: 单元格可以是最多 3-4 周的区域性, 并且有常规媒体更改, 或者没有媒体更改的一周。</li>
</ol>6. 斑点涂料组织培养板/盖玻片 (1 天)
<ol><li>板15µL 的胶原 IV 涂层直接在24良好的阴离子/阳离子涂层组织培养板的中心 (见材料表的细节) 和孵化15分钟在37°c 与 10% CO2。</li>
	<li>在米高梅 hemocytometer 稀释细胞到 2.3 x 105/毫升的细胞计数后。吸入胶原 IV 斑点, 并立即板15µL 细胞悬浮到此部位;这将提供 3.5 x 103单元格/点。孵育 5-10 分钟在37°c 与 10% CO2允许细胞坚持, 增加500µL CO2-平衡 TGF-β2/IL-34/cholesterol 包含生长媒介 (TIC) 温和地对井。</li>
	<li>如果镀在盖玻片, 第一涂层无菌玻璃盖玻片与10µg/毫升的策略 d-赖氨酸 (PDL)在 H 2 o 为 1 H 在 RT, 洗涤 3次与 H 2 O, 并且让干燥在敞篷在紫外光之下。一旦盖玻片干燥, 就按照步骤6.2 中所述, 对盖玻片进行现场电镀。</li>
</ol>7. 用于 RNA/蛋白质分离的电镀
<ol><li>在1天, 涂上24良好阴离子/阳离子涂层组织培养板的整个面积与胶原 IV 涂层和孵育10分钟-1 h 在37°c 与 10% CO2, 然后删除和立即板 3-5 x 104单元格/以及 CO2平衡TIC 媒体。</li>
	<li>在2天, 做好准备后的每一天, 为每个油井执行50% 媒体更改。死细胞往往聚集在井的中心, 所以从那里取媒体。</li>
	<li>每 2-3 天执行50% 媒体更改以维护区域性。如果媒体更改为区域性引入了不需要的变量, 则单元格可以在不发生任何媒体更改的情况下生存大约1周, 而不是3周的定期维护。</li>
</ol>

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提交人声明, 这项研究是在没有任何商业或金融关系的情况下进行的, 这可能被理解为潜在的利益冲突。

由于小胶质细胞作为中枢神经系统的前哨免疫单元, 它们对环境变化有高度的响应能力;因此, 在隔离和区域性8期间, 需要非常小心地最小化细胞内的炎症反应。这在这个协议通过速度和温度完成。保持细胞在冰上或在4°c 只要可能大大减少活化, 所以所有离心步骤发生在4摄氏度, 动物正在灌注冰冷灌注缓冲, 和协议已简化, 以减少组织之间的时间细胞的提取和培养。如果评估新隔离...

作为中枢神经系统实质的巨噬细胞, 小胶质与大量的神经回路和神经胶质信号网络相互作用。通过突触修剪、凋亡细胞间隙和与神经元过程的瞬态相互作用, 它们在大脑的发育和稳态中发挥重要作用1,2。小胶质细胞是早期神经损伤反应者, 将其长而薄的过程扩展到病变部位, 以协调炎症反应并限制出血3,4。胶质形态和功能的变化在急性和慢性中枢神经系统损伤中普遍存在, 小胶质细胞在多种疾病状态下表现出改变的形态学、定位和炎症介质的表达1。人类遗传研究表明, 改变神经退行性疾病风险的突变, 通常主要或完全由小胶质细胞在完整的中枢神经系统表达, 指出小胶质细胞在疾病发病机制或进展中的关键作用5.鉴于他们在伤病和疾病中的突出地位, 进一步了解胶质生物学是发展新的治疗方法的一个高度优先事项。
对胶质生物学认识的许多重要进展, 都是通过推断其他巨噬细胞的研究中发现的技术和机制, 包括培养方法, 基因表达谱, 以及功能的定义。/形态状态。虽然广义巨噬细胞功能经常在中枢神经系统的景观中以惊人的方式发挥出来, 小胶质是他们自己高度专业化, 展示了分支形态学和独特的基因表达签名, 使他们除了其他组织巨噬细胞6。小胶质细胞具有与大多数其他组织巨噬菌不同的血统;他们殖民中枢神经系统在早期胚胎的原始造血和自我更新的整个生命, 独立的贡献从明确的造血7。成年小胶质细胞的完全成熟基因表达签名直到第二个产后周8才实现。来自周围组织的环境提示在口述组织特异性巨噬细胞特征6中起着重要作用, 在中枢神经系统中, 它包括受血脑屏障9所赋予的血液传播因子的有限接触。
充分理解胶质对中枢神经系统稳态和疾病的贡献的一个障碍是综述在体外发现的成熟小胶质细胞的特殊性质的困难, 即在体内的纯化电池为。许多方法已经发展, 以分离和培养完整的小胶质细胞, 但大多数方法依赖于血清, 以支持生存。我们已经表明, 血清, 这是一个固有的变量试剂含有大量的生物活性分子, 是特别成问题, 当与小胶质细胞, 因为它促进变形形态学, 增加增殖, 并增加的吞噬功能9经常看到体内当小胶质细胞暴露于血脑屏障中断后的血液传播因素。根据这些指标, 血清暴露的细胞在损伤或疾病状态下类似小胶质, 但当小胶质体在含有 CSF-1 (或 IL-34)、TGF β、胆固醇和亚硒酸的定义生长培养基中培养时, 这种改变就会减少。
本议定书提供了有关在无血清条件下培养幼鼠小胶质细胞的细节, 与最近发表的工作9有关。该协议已为产后 21-30 (P21-P30) 的大鼠进行了简化, 但可用于将小胶质细胞从任何年龄的老鼠和老鼠中分离出来, 尽管产量和总体生存能力将因动物的种类和年龄而异。当使用稍未成熟的小胶质细胞 (〜 P9) 时, 最大产量和最佳生存能力得到实现, 其产量和活力逐渐减少到成年动物的某些较低水平。小胶质细胞也可以从老鼠身上分离出来, 但我们发现, 与无血清培养的小鼠细胞相比, 鼠体细胞显示出的分支形态的产量、活力和复杂性显著提高。年龄大于 P50 的动物没有被这个协议评估。此 immunopanning 隔离程序已经过优化, 以最小化在隔离过程中胶质转录剖面的变化, 并最大限度地提高细胞的下游生存能力。使用这些技术和媒体配方, 高活力的初级文化可以持续数周。在这些条件下培养的小胶质细胞具有高度分支的形态学, 包括快速延长和缩回过程, 以及相对较低的增殖速率。我们强调了血清暴露对这些特性的意义, 并讨论了该方法相对于其他方法的优缺点。

<table><tbody><tr><td>Rat: Sprague-Dawley</td><td>Charles River</td><td>Cat# 400</td><td></td></tr><tr><td>mouse anti-rat CD11b monoclonal (clone OX42)</td><td>Bio-Rad</td><td>Cat# MCA275R</td><td>Panning: 1:1,000; Staining: 1:500</td></tr><tr><td>Goat polycolonal anti-Iba1</td><td>Abcam</td><td>Cat# AB5076</td><td>Staining: 1:500</td></tr><tr><td>Rabbit polyclonal anti-Ki67</td><td>Abcam</td><td>Cat# AB15580</td><td>Staining: 1:500</td></tr><tr><td>Alexa Fluor Donkey anti-mouse 594</td><td>Invitrogen</td><td>Cat# 11055</td><td>Staining: 1:500</td></tr><tr><td>Alexa Fluor Donkey anti-goat 488</td><td>Invitrogen</td><td>Cat# A-21203</td><td>Staining: 1:500</td></tr><tr><td>Alexa Fluor Donkey anti-Rabbit 647</td><td>Invitrogen</td><td>Cat# A-31573</td><td>Staining: 1:500</td></tr><tr><td>Triton-X (detergent in ICC staining)</td><td>Thermo Fisher</td><td>Cat# 28313</td><td></td></tr><tr><td>Heparan sulfate</td><td>Galen Laboratory Supplies</td><td>Cat# GAG-HS01</td><td></td></tr><tr><td>Heparin</td><td>Sigma&amp;nbsp;</td><td>Cat# M3149</td><td></td></tr><tr><td>Peptone from milk solids</td><td>Sigma</td><td>Cat# P6838</td><td></td></tr><tr><td>TGF-&amp;beta;2</td><td>Peprotech</td><td>Cat# 100-35B</td><td></td></tr><tr><td>Murine IL-34</td><td>R&amp;amp;D Systems</td><td>Cat# 5195-ML/CF</td><td></td></tr><tr><td>Ovine wool cholesterol</td><td>Avanti Polar Lipids</td><td>Cat# 700000P</td><td></td></tr><tr><td>Collagen IV</td><td>Corning&amp;nbsp;</td><td>Cat# 354233</td><td></td></tr><tr><td>Oleic acid</td><td>Cayman Chemicals&amp;nbsp;</td><td>Cat# 90260</td><td></td></tr><tr><td>11(Z)Eicosadienoic (Gondoic) Acid</td><td>Cayman Chemicals&amp;nbsp;</td><td>Cat# 20606</td><td></td></tr><tr><td>Calcein AM dye</td><td>Invitrogen</td><td>Cat# C3100MP</td><td></td></tr><tr><td>Ethidium homodimer-1</td><td>Invitrogen</td><td>Cat# E1169</td><td></td></tr><tr><td>DNaseI</td><td>Worthington</td><td>Cat# DPRFS</td><td></td></tr><tr><td>Percoll PLUS</td><td>GE Healthcare</td><td>Cat# 17-5445-02</td><td></td></tr><tr><td>Trypsin</td><td>Sigma</td><td>Cat# T9935</td><td></td></tr><tr><td>DMEM/F12</td><td>Gibco</td><td>Cat# 21041-02</td><td></td></tr><tr><td>Penicillin/ Streptomycin</td><td>Gibco</td><td>Cat# 15140-122</td><td></td></tr><tr><td>Glutamine</td><td>Gibco</td><td>Cat# 25030-081</td><td></td></tr><tr><td>N-acetyl cysteine&amp;nbsp;</td><td>Sigma</td><td>Cat# A9165</td><td></td></tr><tr><td>Insulin</td><td>Sigma</td><td>Cat# 16634</td><td></td></tr><tr><td>Apo-transferrin&amp;nbsp;</td><td>Sigma</td><td>Cat# T1147</td><td></td></tr><tr><td>Sodium selenite</td><td>Sigma</td><td>Cat# S-5261</td><td></td></tr><tr><td>DMEM (high glucose)</td><td>Gibco</td><td>Cat# 11960-044</td><td></td></tr><tr><td>Dapi Fluoromount-G</td><td>Southern Biotech</td><td>Cat# 0100-20&amp;nbsp;</td><td></td></tr><tr><td>Poly-D-Lysine&amp;nbsp;</td><td>Sigma</td><td>Cat# A-003-E</td><td></td></tr><tr><td>Primaria Plates&amp;nbsp;</td><td>VWR</td><td>Cat# 62406-456</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Stock reagents&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Apo-transferrin</td><td></td><td></td><td>Reconstitution: 10 mg/mL in PBS&lt;br/&gt; Concentration used: 1:100&lt;br/&gt; Storage: -20&amp;deg;C</td></tr><tr><td>N-acetyl cysteine</td><td></td><td></td><td>Reconstitution: 5 mg/mL in H2O&lt;br/&gt; Concentration used: 1:1,000&lt;br/&gt; Storage: -20&amp;deg;C</td></tr><tr><td>Sodium selenite</td><td></td><td></td><td>Reconstitution: 2.5 mg/mL in H2O&lt;br/&gt; Concentration used: 1:25,000&lt;br/&gt; Storage: -20&amp;deg;C</td></tr><tr><td>Collagen IV</td><td></td><td></td><td>Reconstitution: 200 &amp;mu;g/mL in PBS&lt;br/&gt; Concentration used: 1:100&lt;br/&gt; Storage: -80&amp;deg;C</td></tr><tr><td>TGF-b2</td><td></td><td></td><td>Reconstitution: 2 mg/mL in PBS&lt;br/&gt; Concentration used: 1:1,000&lt;br/&gt; Storage: -20&amp;deg;C</td></tr><tr><td>IL-34</td><td></td><td></td><td>Reconstitution: 200 &amp;mu;g/mL in PBS&lt;br/&gt; Concentration used: 1:1,000&lt;br/&gt; Storage: -80&amp;deg;C</td></tr><tr><td>Ovine wool cholesterol</td><td></td><td></td><td>Reconstitution: 1.5 mg/mL in 100% ethanol&lt;br/&gt; Concentration used: 1:1,000&lt;br/&gt; Storage: -20&amp;deg;C</td></tr><tr><td>Heparan sulfate</td><td></td><td></td><td>Reconstitution: 1 mg/mL in H2O&lt;br/&gt; Concentration used: 1:1,000&lt;br/&gt; Storage: -20&amp;deg;C</td></tr><tr><td>Oleic acid/Gondoic acid</td><td></td><td></td><td>Reconstitution: Gondoic: 0.001 mg/mL; Oleic: 0.1 mg/mL in 100% ethanol&lt;br/&gt; Concentration used: 1:1,000&lt;br/&gt; Storage: -20&amp;deg;C</td></tr><tr><td>Heparin</td><td></td><td></td><td>Reconstitution: 50 mg/mL in PBS&lt;br/&gt; Concentration used: 1:100&lt;br/&gt; Storage: -20&amp;deg;C</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Solutions&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Perfusion Buffer</td><td></td><td></td><td>Recipe: 50 &amp;mu;g/mL heparin in DPBS++ (PBS with Ca++ and Mg+ +)&lt;br/&gt; Comments: Use when ice-cold</td></tr><tr><td>Douncing Buffer</td><td></td><td></td><td>Recipe: 200 &amp;mu;L of 0.4% DNaseI in 50 mL of DPBS++&lt;br/&gt; Comments: Use when ice-cold</td></tr><tr><td>Panning Buffer</td><td></td><td></td><td>Recipe: 2 mg/mL of peptone from milk solids in DPBS++</td></tr><tr><td>Microglia Growth Medium (MGM)</td><td></td><td></td><td>Recipe: DMEM/F12 containing 100 units/mL penicillin, 100 &amp;mu;g/mL streptomycin, 2 mM glutamine, 5 &amp;mu;g/mL N-acetyl cysteine, 5 &amp;mu;g/mL insulin, 100 &amp;mu;g/mL apo-transferrin, and 100 ng/mL sodium selenite&lt;br/&gt; Comments: Use ice-cold MGM to pan microglia off of immnopanning dish.</td></tr><tr><td>Collagen IV Coating</td><td></td><td></td><td>Recipe: MGM containing 2 &amp;mu;g/mL collagen IV</td></tr><tr><td>Myelin Seperation Buffer</td><td></td><td></td><td>Recipe: 9 mL Percoll PLUS, 1 mL 10x PBS without Ca++ and Mg++, 9 &amp;mu;L 1 M CaCl2, 5 &amp;mu;L 1 M MgCl2&lt;br/&gt; Comments: Mix well after the addition of&amp;nbsp; CaCl2 and MgCl2</td></tr><tr><td>TGF-b2/IL-34/Cholesterol containing growth media (TIC)&amp;nbsp;</td><td></td><td></td><td>Recipe: MGM containing human 2 ng/mL TGF-b2, 100 ng/mL murine IL-34, 1.5 &amp;mu;g/mL ovine wool cholesterol, 10 &amp;mu;g/mL heparan sulfate, 0.1 &amp;mu;g/ml oleic acid, and 0.001 &amp;mu;g/ml gondoic acid&lt;br/&gt; Comments: Make sure to add cholesterol to media warmed to 37 &amp;deg;C and do not add more than 1.5 &amp;mu;g/mL or it will precipitate out. Do not filter cholesterol-containing media. Equilibrate TIC media with 10% CO2 for 30 min- 1 hr before adding to cells to insure optimal pH.</td></tr></tbody></table>

isolation and culture of rodent microglia to promote a dynamic ramified morphology in serum-free medium

小胶质细胞代表了 5-10% 的所有中枢神经系统 (CNS) 单元, 并越来越引起关注, 因为他们的贡献在发展, 稳态和疾病。尽管巨噬细胞已经被详细研究了数十年, 但小胶质细胞, 中枢神经系统的组织驻留巨噬菌的特殊特征仍然很大程度上是神秘的, 部分原因是对成熟胶质的重述能力的限制。文化中的属性。在这里, 我们说明了一个简单的程序, 快速分离的纯小胶质细胞从成熟的啮齿动物大脑。我们还描述了无血清的文化条件, 支持高水平的胶质生存时间。在这些定义中等条件下培养的小胶质细胞具有精心的分支过程和动态监测行为。我们说明了血清暴露对小胶质细胞的一些影响, 并讨论了这些无血清的文化与血清暴露的文化和小胶质酶在体内的比较。

本协议描述了一种从幼年大鼠中培养高纯度分支小胶质细胞的方法。类似的结果可以得到使用不成熟, 围产期和成年动物, 如下面所述。由于细胞隔离通常包括微妙的细微差别和许多细胞死亡的机会, 我们使用质量控制检查站来帮助确定在各个步骤的成功。我们通常在隔离协议 (图 1A) 的多个步骤中使用 hemocytometer 监视单元格悬浮。成功的隔离应该产生...

Watch this Scientific Journal Video about Isolation and Culture of Rodent Microglia to Promote a Dynamic Ramified Morphology in Serum-free Medium at JoVE.com

Isolation and Culture of Rodent Microglia to Promote a Dynamic Ramified Morphology in Serum-free Medium

由于缺乏对成熟的体内小胶质细胞的特性进行重述的胶质文化模型, 胶质的详细理解的努力受到了阻碍。该协议描述了一种隔离和培养方法, 旨在维持高分支成熟大鼠小胶质细胞在确定培养基条件下的健壮生存。

鼠小胶质细胞的分离培养促进无血清培养基动态分支形态学的研究

Microglia represent 5 - 10% of all central nervous system (CNS) cells and are increasingly drawing attention due to their contributions during ...

isolation-culture-rodent-microglia-to-promote-dynamic-ramified

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

15.8K 次观看.  Stanford University. 由于缺乏对成熟的体内小胶质细胞的特性进行重述的胶质文化模型, 胶质的详细理解的努力受到了阻碍。该协议描述了一种隔离和培养方法, 旨在维持高分支成熟大鼠小胶质细胞在确定培养基条件下的健壮生存。

视频: Isolation and Culture of Rodent Microglia to Promote a Dynamic Ramified Morphology in Serum-free Medium

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.

Isolating primary microglia from the cellular heterogeneity of the brain is essential to investigate their role in both physiological and pathological conditions. This protocol describes a mechanical isolation and mixed cell culture technique that provides high yield and high purity, viable primary microglial cells for in vitro study and downstream applications.

初级小胶质细胞的分离，从新生大鼠脑组织胶质细胞混合文化

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

科研

免疫与感染

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

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure for isolation of microglia from neonatal murine cortices by mechanical agitation with a rotary shaker. This microglia isolation method yields highly pure cortical microglia that exhibit morphological and functional characteristics indicative of quiescent microglia in normal, nonpathological conditions in vivo. This procedure also preserves the microglial immunophenotype and biochemical functionality as demonstrated by the induction of morphological changes, nuclear translocation of the p65 subunit of NF-&#954;B (p65), and secretion of the hallmark proinflammatory cytokine, tumor necrosis factor-&#945; (TNF-&#945;), upon lipopolysaccharide (LPS) and Pam3CSK4 (Pam) challenges. Therefore, the present isolation procedure preserves the immunophenotype of both quiescent and activated microglia, providing an experimental method of investigating microglia biology in ex vivo conditions.

One key to successful investigation of microglial biology is the preservation of microglial immunofunction ex vivo during isolation from CNS tissue. Isolating microglia via rotary shaking results in highly pure and immunofunctional cell cultures as assessed by fluorescent imaging, immunocytochemistry, and ELISA following microglia activation with the proinflammatory stimuli lipopolysaccharide (LPS) and Pam3CSK4 (Pam).

皮层小胶质细胞的分离与保存免疫表型和功能从新生儿鼠

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

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.

密度梯度离心法分离小鼠原发胶质细胞的研究

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

神经科学

Dissection and Isolation of Murine Glia from Multiple Central Nervous System Regions

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine neonates for the isolation of glial subpopulations. The purpose of the procedure is to dissociate microglia, oligodendrocyte progenitor cells (OPCs), and astrocytes from cortical, cerebellar, brainstem, and spinal cord tissue to facilitate further in vitro analysis. The CNS region isolation procedures allow for the determination of regional heterogeneity among glia in multiple cell culture systems. Rapid CNS region isolation is performed, followed by the mechanical removal of meninges to prevent meningeal cell contamination of glia. This protocol combines gentle tissue dissociation and plating on a specified matrix designed to preserve cell integrity and adherence. Isolating mixed glia from multiple CNS regions provides a comprehensive analysis of potentially heterogenous glia while maximizing the use of individual experimental animals. Additionally, following dissociation of regional tissue, mixed glia are further divided into multiple cell types including microglia, OPCs, and astrocytes for use in either single cell type, cell culture plate inserts, or co-culture systems. Overall, the demonstrated techniques provide a comprehensive protocol of broad applicability for careful dissection of four individual CNS regions from murine neonates and includes methods for the isolation of three individual glia cell types to examine regional heterogeneity in any number of in vitro cell culture systems or assays.

Here we present a protocol for in vitro isolation of multiple glial cell populations from a mouse CNS. This method allows for the segregation of regional microglia, oligodendrocyte precursor cells, and astrocytes to study the phenotypes of each in a variety of culture systems.

从多个中枢神经系统区域解剖和分离小鼠神经胶质细胞

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine ...

Obtaining Human Microglia from Adult Human Brain Tissue

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining homeostasis, and during infection or injury. Several independent research groups have highlighted the central role that microglia play in autoimmune diseases, autoinflammatory syndromes and cancers. The activation of microglia in some neurological diseases may directly participate in pathogenic processes. Primary microglia are a powerful tool to understand the immune responses in the brain, cell-cell interactions and dysregulated microglia phenotypes in disease. Primary microglia mimic in vivo microglial properties better than immortalized microglial cell lines. Human adult microglia exhibit distinct properties as compared to human fetal and rodent microglia. This protocol provides an efficient method for isolation of primary microglia from adult human brain. Studying these microglia can provide critical insights into cell-cell interactions between microglia and other resident cellular populations in the CNS including, oligodendrocytes, neurons and astrocytes. Additionally, microglia from different human brains may be cultured for characterization of unique immune responses for personalized medicine and a myriad of therapeutic applications.

This protocol is an efficient, cost effective and robust method of isolating primary microglia from live, adult, human brain tissue. Isolated primary human microglia can serve as a tool for studying cellular processes in homeostasis and disease.

从成人人类脑组织获取人类微胶质

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining ...

Magnetic Isolation of Microglial Cells from Neonate Mouse for Primary Cell Cultures

Microglia, as brain resident macrophages, are fundamental to several functions, including response to environmental stress and brain homeostasis. Microglia can adopt a large spectrum of activation phenotypes. Moreover, microglia that endorse pro-inflammatory phenotype is associated with both neurodevelopmental and neurodegenerative disorders. In vitro studies are widely used in research to evaluate potential therapeutic strategies in specific cell types. In this context studying microglial activation and neuroinflammation in vitro using primary microglial cultures is more relevant than microglial cell lines or stem-cell-derived microglia. However, the use of some primary cultures might suffer from a lack of reproducibility. This protocol proposes a reproducible and relevant method of magnetically isolating microglia from neonate pups. Microglial activation using several stimuli after 4 h and 24 h by mRNA expression quantification and a Cy3-bead phagocytic assay is demonstrated here. The current work is expected to provide an easily reproducible technique for isolating physiologically relevant microglia from juvenile developmental stages.

Primary microglia cultures are commonly used to evaluate new anti-inflammatory molecules. The present protocol describes a reproducible and relevant method to magnetically isolate microglia from neonate pups.

从新生小鼠中磁分离小胶质细胞用于原代细胞培养

Microglia, as brain resident macrophages, are fundamental to several functions, including response to environmental stress and brain homeostasis. ...

Isolation of Microglia from the Cortex of an Adult Mouse Brain

Source: Zhou, L., et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/60347">Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing.</a> J. Vis. Exp. (2019)
The video demonstrates a technique for isolating microglia from the cortex of an adult mouse brain. First, the cortex undergoes segmentation and homogenization to remove neurons and other glial cells, preserving microglia. Following that, the cell suspension is exposed to anti-myelin magnetic microbeads and a magnetic field, effectively separating microglia and myeloid cells from myelin debris and cells containing myelin.

从成年小鼠大脑的皮层中分离小胶质细胞

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Mechanical Tissue Dissociation
...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Primary Neuronal Culture

Selectively Removing Microglia from a Rat Cerebellar Granule Neuron Culture

Source: Jebelli, J., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/52983">Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester.</a> J. Vis. Exp. (2015)
This video demonstrates a technique for the selective removal of microglia from a primary rat neuronal culture. Cerebellar granule neurons are treated with an antimetabolite to eliminate proliferating glial cells. Additionally, they are treated with lysosome-targeting agents to deplete microglia from the culture.

从大鼠小脑颗粒神经元培养物中选择性去除小胶质细胞

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparation of Instruments, ...

Co-culture and Interaction Studies

Isolation and Culture of Microglia from Rat Brains using Serum-Free Media

Source: Collins, H. Y., et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/57122">Isolation and Culture of Rodent Microglia to Promote a Dynamic Ramified Morphology in Serum-free Medium.</a> J. Vis. Exp. (2018)
This video demonstrates a method for isolating and culturing microglia from rat brains. It entails separating microglia from other neuronal and non-neuronal cells using plates coated with a monoclonal antibody specific to microglia.

使用无血清培养基从大鼠脑中分离和培养小胶质细胞

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Prepare a Petri Dish for CD11b ...

Magnetic Isolation of Microglia from Mouse Pup Brains

Source: Bokobza, C. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/62964">Magnetic Isolation of Microglial Cells from Neonate Mouse for Primary Cell Cultures.</a> J. Vis. Exp. (2022)
In this video, we describe a protocol for isolating CD11b cells, including microglia, from mouse pup brains using magnetic-activated cell sorting. The brain tissues are homogenized to obtain single-cell suspensions, which are incubated with anti-CD11b antibody-coated microbeads, followed by a magnetic column-based separation to purify the CD11b+ cell population.

从小鼠幼崽大脑中磁性分离小胶质细胞

Source: Bokobza, C. et al., Magnetic Isolation of Microglial Cells from Neonate Mouse for Primary Cell Cultures. J. Vis. Exp. (2022)
In this video, we ...