JP 感谢加拿大自然科学与工程研究委员会和萨斯喀彻温大学医学院的资金支持。YD 感谢萨斯喀彻温大学医学院启动基金、加拿大自然科学与工程研究委员会发现补助金 （RGPIN-2023-03659）、MS Canada 催化剂补助金 （1019973）、萨斯喀彻温省健康研究基金会建立补助金 （6368） 和 Brain Canada Foundation 加拿大脑研究未来领袖补助金的资金支持。 图 1A、 图 2A 和 图 3A 是使用 BioRender.com 创建的。

本研究中使用的所有动物均经萨斯喀彻温大学动物护理委员会 （UACC） 和加拿大动物护理委员会 （CCAC） 批准进行饲养和处理。本研究使用怀孕 CD1 小鼠出生后 0-2 天 CD1 雄性和雌性小鼠以及胚胎第 16-18 天 （E16-18） 胚胎。材料 表中列出了试剂和所用设备的详细信息。
1. 原代小胶质细胞培养
注意：对混合神经胶质细胞和神经元培养物进行计时至关重要，以便小胶质细胞在将神经元接种到 96 孔板后 2 天内成熟并准备好收获。
<ol><li>制备<ol><li>预热完全神经胶质细胞培养基，包括高葡萄糖的 Dulbecco 改良 Eagle 培养基 （DMEM），其中补充有 10% 热灭活胎牛血清、50 U/mL 青霉素/链霉素和 2.92 mg/mL L-谷氨酰胺、1 mM 丙酮酸钠、1x 非必需氨基酸和 20 mM HEPES，在 37 °C 水浴中。</li>
		<li>向每个 T-75 培养瓶中加入 5 mL 10 μg/mL 聚-L-鸟氨酸 （PO） 氢溴酸盐。轻轻旋转培养瓶，确保 PO 完全覆盖培养瓶底部。在 37 °C 下在 5% CO2 培养箱中孵育至少 1 小时。 注：此包被步骤是促进细胞粘附到 T-75 培养瓶上所必需的。</li>
	</ol></li>
	<li>从 P0-P2 新生小鼠中分离大脑 注意：对所有解剖工具进行消毒并保持试剂的无菌性。确保全程遵守无菌技术。<ol><li>在开始解剖之前，在将要进行解剖的地方用 70% 乙醇或消毒喷雾喷洒台面。设置解剖显微镜并将培养皿置于显微镜下。将 20 mL 的 Leibovitz 的 L-15 培养基倒入培养皿中。不使用显微镜时，请保持培养皿的盖子打开，以减少污染。</li>
		<li>放下几层纸巾，用 70% 乙醇彻底喷洒纸巾。准备解剖工具（解剖剪刀、一对微型镊子和组织镊子），用 70% 乙醇彻底喷洒，然后将工具放在纸巾上。</li>
		<li>将出生后 0-2 天的小鼠转移到纸巾上，并用 70% 乙醇彻底喷洒小鼠。使用解剖剪刀将小鼠斩首（遵循机构批准的方案）。</li>
		<li>轻轻握住头部，在颅骨上做一个从颈部到鼻子的中线切口。用组织镊子将头骨向后剥开，露出大脑。</li>
		<li>使用组织镊子，将镊子插入大脑下方，轻轻地将大脑舀出，然后将大脑从头部提起。立即将大脑放入含有 20 mL Leibovitz's L-15 培养基的培养皿中。</li>
		<li>在解剖显微镜下，使用一对微型镊子小心地去除脑干和脑膜。将大脑收集在含有 5 mL Leibovitz's L-15 培养基的 50 mL 锥形管中。</li>
	</ol></li>
	<li>脑解离、接种和维持混合胶质细胞培养 注：在生物安全柜中执行所有后续步骤。<ol><li>用无菌的一次性手术刀刀片将大脑切成约 1 毫米2 的小块。小心地将切碎的大脑转移到新的 50 mL 无菌试管中。</li>
		<li>向切碎的大脑中加入终浓度为 0.25% 胰蛋白酶的胰蛋白酶，并将组织在 37 °C 水浴中孵育 20 分钟。每 2-3 分钟，轻轻倒置试管以混合溶液，以帮助组织解离。</li>
		<li>从 5% CO2 培养箱中，将含有 5 mL PO 的 T-75 培养瓶转移到生物安全柜中。丢弃 PO 并用无菌 1x 磷酸盐缓冲溶液 （PBS） 冲洗培养瓶 3 次。确保彻底冲洗培养瓶，因为过量的 PO 对细胞有毒。</li>
		<li>将消化的组织从 37 °C 水浴转移到生物安全柜中。加入 5 mL 完全神经胶质培养基以中和胰蛋白酶。使用 10 mL 移液器轻轻混合组织悬液。</li>
		<li>将无菌 70 μm 尼龙网孔池过滤器放在无菌 50 mL 锥形管上，倒入大约 5 mL L-15 润湿网孔。小心地将细胞悬液倒入细胞过滤器中。从无菌 1 mL 注射器中取出柱塞，使用柱塞将组织研磨成网片以去除组织团块，并产生匀浆细胞悬液。</li>
		<li>定期将大约 5 mL 的 Leibovitz L-15 培养基倒入 70 μm 尼龙网中，然后用 1 mL 注射器柱塞继续研磨网上的组织。当网状物上几乎没有可见组织时，丢弃网状物并将细胞悬液放在一边。</li>
		<li>通过轻轻上下移液来混合细胞悬液。向每个培养瓶中加入等体积的含有 1-2 个脑的细胞悬液和神经胶质细胞培养基，最终体积为 15 mL。</li>
		<li>将细胞在 37 °C 下在 5% CO2 培养箱中孵育过夜。第二天，用无菌 1x PBS 洗涤培养瓶两次，以去除未附着的细胞和碎片。</li>
		<li>向培养物中加入 15 mL 新鲜完全神经胶质细胞培养基，并在 37 °C 下在 5% CO2 培养箱中孵育。3 天后，用 10 mL 新鲜神经胶质细胞培养基更换一半的培养基，其中补充有 40 ng/mL 巨噬细胞集落刺激因子 （MCSF）。 注意：此后，每 3 天用 10 mL 新鲜神经胶质细胞培养基替换一半的培养基，并补充 40 ng/mL 的 MCSF。在收获小胶质细胞之前，培养物维持 8-10 天。</li>
	</ol></li>
</ol>2. 原代神经元培养
<ol><li>制备<ol><li>解冻来自 B-27 plus 神经元培养系统的 B-27 plus 补充剂。来自 B-27 的 neurobasal plus 培养基加神经元培养系统、1x Hank 平衡盐溶液 （HBSS） 和热灭活胎牛血清 （FBS），在 37 °C 下在水浴中。</li>
		<li>用 5 mL 10 μg/mL PO 包被 T-25 培养瓶，并在 37 °C 下孵育至少 1 小时。 准备用于解剖的器械和用品。 注：该包被步骤有助于细胞粘附到 T-25 培养瓶上。</li>
	</ol></li>
	<li>从 E16-E18 小鼠胚胎中分离大脑 注意：对所有解剖工具进行消毒并保持试剂的无菌性。确保全程遵守无菌技术。<ol><li>在开始解剖之前，在将要进行解剖的地方用 70% 乙醇或消毒喷雾喷洒台面。设置解剖显微镜并将培养皿置于显微镜下。将 20 mL 的 1x HBSS 倒入培养皿中。不使用显微镜时，请保持培养皿的盖子打开，以减少污染。</li>
		<li>放下几层纸巾，用 70% 乙醇彻底喷洒纸巾。准备解剖工具（弹簧剪刀、一对微型镊子和组织镊子），用 70% 乙醇彻底喷洒，然后将工具放在纸巾上。</li>
		<li>在妊娠第 16-18 天之间使用异氟醚麻醉怀孕的小鼠，并通过宫颈脱位对小鼠实施安乐死（遵循机构批准的方案）。</li>
		<li>将鼠标仰卧在一块浸泡了 70% 乙醇的纸巾上。用 70% 乙醇消毒腹部区域。用组织镊子提起下腹部的皮肤，并用解剖剪刀从该点到下肋骨做一个 V 形切口。</li>
		<li>用组织镊子抓住含有胚胎的子宫角，轻轻地从腹腔中取出胚胎。用 70% 乙醇对含有胚胎的子宫角进行短暂消毒。</li>
		<li>一次从其单个囊中解剖一个胚胎。使用弹簧剪刀，在颅骨顶部创建一个中线切口，从脖子后部开始到鼻子。然后，将头骨从切口部位提起，露出大脑。用组织镊子轻轻舀出大脑，然后将大脑转移到含有 15 mL 1x HBSS 的 10 厘米培养皿中。</li>
		<li>在解剖显微镜下，用一对微型镊子小心地去除脑干和脑皮层两侧的脑膜。将脑皮层转移到含有 10 mL 1x HBSS 的 50 mL 锥形管中，并置于冰上。</li>
	</ol></li>
	<li>细胞培养中大脑解离和皮层神经元接种 注：在生物安全柜中执行所有后续步骤。<ol><li>用无菌一次性手术刀刀片在 50 mL 锥形管中将大脑切成约 1 毫米2 块。将切碎的大脑转移到新的无菌 50 mL 锥形管中。</li>
		<li>将胰蛋白酶加入试管中，终浓度为 0.25%。将试管浸入 35-38 °C 热水浴中 15 分钟，然后每 2-3 分钟轻轻混合试管以匀浆组织。</li>
		<li>将管子从热水浴中取出。轻轻上下移液组织悬液以进一步解离细胞，然后向匀浆悬浮液中加入 0.5 mL 热灭活的 FBS 以中和胰蛋白酶活性。避免气泡，以尽量减少对神经元细胞的毒性。</li>
		<li>将无菌 70 μm 尼龙网状细胞过滤器放在无菌 50 mL 锥形管上，并加入大约 5 mL 的 neurobasal 培养基润湿网状细胞。通过 70 μm 尼龙网状细胞过滤器过滤细胞悬液。使用 1 mL 注射器柱塞，将组织轻轻研磨到细胞过滤器中。定期倒入大约 5 mL 的 1x HBSS，同时继续研磨以清洗细胞过滤器。</li>
		<li>将锥形管在 4 °C 下以 300 x g 离心 5 分钟。 在此期间，将 Neurobasal plus 培养基中的 B-27 plus 补充剂稀释至最终浓度的 1 倍，以用作完整的 neurobasal 培养基。</li>
		<li>丢弃 T-25 烧瓶中的 PO，并用 5 mL 的 1x PBS 冲洗烧瓶 3 次。</li>
		<li>小心倒出以去除上清液，并通过轻轻上下吹打 2-3 mL 完全神经基底培养基来重悬细胞沉淀。用血细胞计数器和台盼蓝测定细胞浓度和总细胞数。预计每个胚胎最多有 1.0 x 107 个细胞。</li>
		<li>将细胞接种在 PO 包被的 T-25 培养瓶中，每个培养瓶约 2.0 x 107 个细胞，溶于 5 ml 完全神经基底培养基中。将细胞培养物在 37 °C 下在 5% CO2 培养箱中孵育。</li>
		<li>每 2-3 天更换一次半培养基，用新鲜制备的完全神经基底培养基替换培养瓶中的 2.5 mL 培养基。</li>
	</ol></li>
</ol>3. 原代神经元和小胶质细胞的共培养
注：所有后续步骤均应在无菌生物安全柜中完成。
<ol><li>在 96 孔板中接种神经元<ol><li>每孔加入 100 μL 10 μg/mL PO，并在 37 °C 下孵育烧瓶至少 1 小时，以用 PO 包被孔。在接种细胞之前，用 1x PBS 洗涤板 3 次，并在最后一次洗涤后吸出任何剩余的液体。</li>
		<li>在 T-25 培养瓶中生长的神经元在培养 2-5 天后收获用于共培养。用 5 mL 含 0.25% 胰蛋白酶和 1 mg/mL DNase I 的 1x Versene 溶液更换培养基，以从培养瓶中分离神经元。将培养瓶放入培养箱中 5-6 分钟进行消化，每 2-3 分钟轻轻旋转一次培养瓶，以帮助细胞分离。</li>
		<li>从培养箱中取出培养瓶，在显微镜下检查细胞是否脱落。加入 0.5 mL 热灭活的 FBS 以中和胰蛋白酶和 DNase I。将细胞转移到 15 mL 锥形管中。用 5 mL 完全 neurobasal 培养基洗涤培养瓶一次，以最大限度地收集神经元。将所有细胞收集在同一个 15 mL 锥形管中。</li>
		<li>将细胞悬液在 4 °C 下以 300 x g 离心 5 分钟。 弃去上清液，将细胞沉淀轻轻重悬于 2-3 mL 完全神经基底培养基中。</li>
		<li>用血细胞计数器计数细胞，并加入完整的神经基础培养基，以达到 7.5 x 105 个神经元/mL 的最终浓度。将 100 μL 细胞悬液接种到 PO 包被的 96 孔板中的每个孔中，以获得 7.5 x 104 个神经元/孔。预计每个培养瓶的产量为 5-600 万个神经元。</li>
		<li>将神经元在 96 孔板中孵育过夜。第二天向每个孔中加入 100 μL 完全 neurobasal 培养基。 注意：一旦神经元表现出适当的神经突突生长（在 96 孔板中接种后第 3-4 天左右），它们就可以与小胶质细胞共培养。</li>
	</ol></li>
	<li>分离和小胶质细胞-神经元共培养<ol><li>在混合胶质细胞培养 8-10 天之间，用 10 mL 移液管轻轻洗涤每个培养瓶中装有神经胶质细胞培养基的 T-75 培养瓶，收集小胶质细胞，然后将细胞和培养基转移到无菌的 50 mL 锥形管中。用 10 mL 新鲜神经胶质细胞培养基再次洗涤培养瓶，以从培养瓶中分离额外的小胶质细胞并收集培养基。预计每个培养瓶的产量为 7.5 x 105 个小胶质细胞。</li>
		<li>向每个培养瓶中加入 20 mL 补充有 20 ng/mL MCSF 的新鲜神经胶质细胞培养基。如果维持混合胶质细胞培养物，小胶质细胞可以再收获一次或两次。</li>
		<li>将细胞收集物在 4 °C 下以 300 x g 离心 5 分钟。 小心去除上清液，并将沉淀轻轻重悬于 2 mL 完全 neurobasal 培养基中。</li>
		<li>用血细胞计数器和台盼蓝计数细胞。添加完全的 neurobasal 培养基，终浓度为 2.5 x 105 个细胞/mL。</li>
		<li>从 96 孔神经元培养板中，去除 100 μL 神经基底培养基，并加入 100 μL 2.5 x 105 个细胞/mL 细胞悬液，以每孔 7.5 x 104 个神经元接种 2.5 x 104 个小胶质细胞。将培养物在 37 °C 下在 5% CO2 培养箱中孵育过夜，以使小胶质细胞沉淀。共培养物现在可用于下游实验。</li>
	</ol></li>
</ol>

 用于 CNS 相互作用研究的原代小胶质细胞和神经元共培养方案

作者声明没有利益冲突。

本文介绍了一种分离和培养小鼠原代神经元和原代小胶质细胞的方案，随后用于建立小胶质细胞-神经元共培养物，可用于研究小胶质细胞和神经元相互作用如何调节其细胞健康和功能。这种相对简单易行的方法可以为 CNS 中小胶质细胞神经元相互作用的机制和功能结果提供关键见解。
为了实现最佳的共培养，该方案中的几个关键方面需要格外小心。在脑分离期间（步骤 1.2.6 ?...

小胶质细胞是组织驻留的巨噬细胞，可促进中枢神经系统 （CNS） 的免疫监视和稳态1,2,3。它们起源于卵黄囊红髓样祖细胞，在胚胎发育过程中定植于大脑 4,5,6，并通过自我更新（涉及增殖和细胞凋亡）在生物体的整个生命周期中维持7。在稳态时，静息小胶质细胞分化形态并参与组织监测 8,9,10。
小胶质细胞表达许多细胞表面受体，这使它们能够快速响应 CNS 的变化11,12，并在感染或组织损伤的情况下促进炎症反应 12,13,14，以及在神经退行性疾病期间 9,15，如多发性硬化症 （MS）16,17.小胶质细胞还表达各种神经递质和神经肽的受体 18,19,20，这表明它们也可能响应并调节神经元活动 21,22。事实上，小胶质细胞和神经元以各种形式的双向通讯相互作用 8,23，例如由膜蛋白介导的直接相互作用或通过可溶性因子或中间细胞的间接相互作用23,24。
例如，神经元分泌的各种神经递质可以调节小胶质细胞的神经保护或炎症活性 25,26,27。此外，神经元和小胶质细胞之间的直接相互作用有助于将小胶质细胞维持在稳态28。相反，小胶质细胞与神经元的直接相互作用可以塑造神经元回路29 并影响神经元信号转导 30,31,32。由于这些相互作用的破坏会诱导神经元的过度兴奋30 和小胶质细胞反应性33,34，失调的小胶质细胞-神经元相互作用被认为是神经系统疾病的一个促成因素33,35。事实上，精神病23,26 和神经退行性疾病已被描述为表现出功能失调的小胶质细胞-神经元相互作用33。虽然这些观察结果强调了小胶质细胞-神经元通讯在 CNS 中的重要性，但这些相互作用如何调节健康和疾病中小胶质细胞和神经元功能的具体机制相对未知。
在 CNS 等复杂环境中，多种环境因素会影响小胶质细胞-神经元相互作用，从而限制了研究 体内瞬时细胞相互作用的能力。在这里，我们提出了一种 体外 小胶质细胞-神经元共培养系统，可用于研究小胶质细胞和神经元之间的直接细胞相互作用。该方案分别描述了出生后第 0-2 天和胚胎小鼠第 16-18 天之间从新生小鼠皮层产生原代小胶质细胞和神经元。然后将神经元和小胶质细胞在 96 孔板中共培养，用于下游高通量实验。我们之前使用这种方法来证明小胶质细胞吞噬作用保护神经元免受氧化磷脂酰胆碱介导的细胞死亡37，这表明这种方法可以帮助理解小胶质细胞在神经变性和 MS 中的作用。同样，小胶质细胞-神经元共培养也可能有助于研究小胶质细胞-神经元串扰在其他情况下的影响，例如病毒感染38 或神经元损伤和修复39。总体而言， 体外小 胶质细胞-神经元共培养系统使研究人员能够在可操作和受控的环境中研究小胶质细胞-神经元相互作用，这与 体内 模型相辅相成。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 cm Petri dish&#160;</td>
			<td>Fisher&#160;</td>
			<td>07-202-011</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>1x Versene</td>
			<td>Gibco</td>
			<td>15040-066</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Plus Neuronal Culture System&#160;</td>
			<td>Gibco&#160;</td>
			<td>A3653401</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection microscope</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Roche</td>
			<td>11284932001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium (DMEM)</td>
			<td>Gibco</td>
			<td>11960-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum&#160;</td>
			<td>ThermoFisher Sci</td>
			<td>12483-020</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS (10x)</td>
			<td>Gibco</td>
			<td>14065-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Hausser Scientific</td>
			<td>1475</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES&#160;</td>
			<td>ThermoFisher Sci</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>Leibovitz&#8217;s L-15 Medium (1x)</td>
			<td>Fisher Scientific&#160;</td>
			<td>21083027</td>
			<td></td>
		</tr>
		<tr>
			<td>Macrophage colony stimulating factor&#160;</td>
			<td>Peprotech</td>
			<td>315-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Forceps</td>
			<td>RWD</td>
			<td>F11020-11</td>
			<td>Autoclaved/Sterile</td>
		</tr>
		<tr>
			<td>Non-essential amino acids</td>
			<td>Cytiva</td>
			<td>SH3023801</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (10x)</td>
			<td>ThermoFisher Sci</td>
			<td>AM9625</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin Glutamine (100x)</td>
			<td>Gibco</td>
			<td>103780-16</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-ornithine hydrobromide&#160;</td>
			<td>Sigma</td>
			<td>P3655-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate (100 mM)</td>
			<td>Gibco</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Spring scissors</td>
			<td>RWD</td>
			<td>S11008-42</td>
			<td>Autoclaved/Sterile</td>
		</tr>
		<tr>
			<td>Surgical blade</td>
			<td>Feather</td>
			<td>08-916-5D</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>T-25 flasks</td>
			<td>Fisher</td>
			<td>10-126-9</td>
			<td></td>
		</tr>
		<tr>
			<td>T-75 flasks&#160;</td>
			<td>Fisher</td>
			<td>13-680-65</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue forceps</td>
			<td>Codman</td>
			<td>30-4218</td>
			<td>Autoclaved/Sterile</td>
		</tr>
		<tr>
			<td>Tissue scissors</td>
			<td>RWD</td>
			<td>S12052-10</td>
			<td>Autoclaved/Sterile</td>
		</tr>
		<tr>
			<td>Trypan Blue&#160;</td>
			<td>Thermofisher Sci&#160;</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin (2.5%)</td>
			<td>ThermoFisher Sci</td>
			<td>15090046</td>
			<td></td>
		</tr>
		<tr>
			<td>Widefield Immunofluorescence Microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

generating and co-culturing murine primary microglia and cortical neurons

小胶质细胞是中枢神经系统 （CNS） 的组织驻留巨噬细胞，执行支持神经元健康和 CNS 稳态的许多功能。它们是与 CNS 疾病活动相关的免疫细胞的主要群体，采用反应性表型，在慢性神经退行性疾病（如多发性硬化症 （MS））期间可能导致神经元损伤。由于解决小胶质细胞、神经元和其他 CNS 环境因素之间复杂的 体内 相互作用的挑战，小胶质细胞在健康和疾病期间调节神经元功能和存活的不同机制仍然有限。因此，共培养小胶质细胞和神经元的 体外 方法仍然是研究小胶质细胞-神经元相互作用的宝贵工具。在这里，我们提出了一种从小鼠生成和共培养原代小胶质细胞和神经元的方案。具体来说，在出生后 0-2 天之间，从来自新生小鼠脑匀浆的混合胶质细胞培养物 中 分离出小胶质细胞。在胚胎第 16-18 天之间从小鼠胚胎的脑皮层中分离出神经元细胞。 体外 4-5 天后，将神经元细胞接种在 96 孔板中，然后加入小胶质细胞以形成共培养物。仔细的时间安排对于该方案至关重要，因为两种细胞类型都需要达到实验成熟度才能建立共培养。总体而言，这种共培养可用于研究小胶质细胞-神经元相互作用，并且可以提供多种读数，包括免疫荧光显微镜检查、实时成像以及 RNA 和蛋白质检测。

图 1A 显示了小胶质细胞混合胶质细胞培养关键步骤的流程图。总体而言，预计第 1 天会出现稀疏的细胞和过多的细胞碎片（图 1B）。到第 4 天，应观察到细胞数量增加，尤其是贴壁星形胶质细胞的产生，如其细长的形态所示（图 1C）。可以在星形胶质细胞顶部观察到一些小胶质细胞，或者作为漂浮在培养基中的小圆形细胞。到第...

作者聚焦：用于研究疾病条件下小胶质细胞-神经元相互作用的体外共培养模型

该方案描述了从胚胎第 15-16 天从小鼠胚胎中分离的原代神经元细胞和出生后第 1-2 天从新生小鼠大脑产生的原代小胶质细胞建立的小胶质细胞-神经元共培养物。

生成和共培养小鼠原代小胶质细胞和皮质神经元

Microglia are tissue-resident macrophages of the central nervous system (CNS), performing numerous functions that support neuronal health and CNS ...

generating-co-culturing-murine-primary-microglia-cortical

Research

JoVE Journal

Neuroscience

Generating and Co-culturing Murine Primary Microglia and Cortical Neurons

1.5K 次观看.  University of Saskatchewan. 我们的研究旨在研究多发性硬化症等疾病条件下神经元和小胶质细胞之间的相互作用。我们的目标是在体外概括小胶质细胞神经元的相互作用，以更好地了解它们在 CNS 中的功能重要性。我们之前证明，小胶质细胞吞噬作用通过这种共培养保护神经元免受氧化磷脂酰胆碱介导的细胞死亡。虽然还采用了其他共培养系统，但这种混合培养系统能够?...

视频: 作者聚焦：用于研究疾病条件下小胶质细胞-神经元相互作用的体外共培养模型

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

在子宫内的电穿孔其次原发性神经文化为研究基因的功能在皮层神经元的一个子集

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

科研

神经科学

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Nucleofection和胚胎小鼠海马和皮层神经元的原代培养

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

二层共培养小学的大鼠皮层神经元和神经胶质

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

Calcium Phosphate Transfection of Primary Hippocampal Neurons

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells.

原代海马神经元的磷酸钙转染

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this ...

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.

原代小胶质的快速和精细的CD11b电磁隔离带增强的纯度和多功能性

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.

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 ...

Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is important to isolate these cells without changing their gene expression profile. The zebrafish model provides a large number of transgenic fish lines in which specific cell types are labelled; for example neurons in the NBT:DsRed line or macrophages/microglia in the mpeg1:eGFP line. Furthermore, antibodies have been developed to stain specific cells, such as microglia with the 4C4 antibody.
Here, we describe the isolation of neurons, macrophages and microglia from larval zebrafish brains. Central to this protocol is the avoidance of an enzymatic tissue digestion at 37 &#176;C, which could modify cellular profiles. Instead a mechanical system of tissue homogenization at 4 &#176;C is used. This protocol entails homogenization of brains into cell suspension, their immuno-staining and the isolation of neurons, macrophages and microglia by FACS. Afterwards, we extracted RNA from those cells and evaluated their quality/quantity. We managed to obtain RNA of high quality (RNA Integrity Number (RIN) &#62; 7) to perform qPCR on macrophages/microglia and neurons, and transcriptomic analysis on microglia. This approach enables a better characterization of these cells, as well as a clearer understanding about their role in development and pathologies.

We present a protocol to isolate neurons, macrophages and microglia from larval zebrafish brains under physiological and pathological conditions. Upon isolation, RNA is extracted from these cells to analyze their gene expression profile. This protocol allows for the collection of high-quality RNA for performing downstream analysis like qPCR and transcriptomics.

斑马鱼脑幼虫神经元、巨噬细胞和小胶质的分离和 RNA 提取

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is ...

Use of Pre-Assembled Plastic Microfluidic Chips for Compartmentalizing Primary Murine Neurons

Microfabricated methods to compartmentalize neurons have become essential tools for many neuroscientists. This protocol describes the use of a commercially available pre-assembled plastic chip for compartmentalizing cultured primary rat hippocampal neurons. These plastic chips, contained within the footprint of a standard microscope slide, are compatible with high-resolution, live, and fluorescence imaging. This protocol demonstrates how to retrograde label neurons via isolated axons using a modified rabies virus encoding a fluorescent protein, create isolated microenvironments within one compartment, and perform axotomy and immunocytochemistry on-chip. Neurons are cultured for &gt;3 weeks within the plastic chips, illustrating the compatibility of these chips for long-term neuronal cultures.

This protocol describes the use of plastic chips to culture and compartmentalize primary murine neurons. These chips are preassembled, user-friendly, and compatible with high-resolution, live, and fluorescence imaging. This protocol describes how to plate rat hippocampal neurons within these chips and perform fluidic isolation, axotomy and immunostaining.

预组装塑料微流体芯片在原生小鼠神经元分割中的应用

Microfabricated methods to compartmentalize neurons have become essential tools for many neuroscientists. This protocol describes the use of a ...

Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-E16 Sprague Dawley Rat Embryo

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have a robust in vitro model that can be exploited for various neurobiology studies. Though primary neuron cultures (i.e., long-term hippocampal cultures) have provided scientists with models, it does not yet represent the complexity of brain network completely. In the wake of these limitations, a new model has emerged using neurospheres, which bears a closer resemblance to the brain tissue. The present protocol describes the plating of high and low densities of mixed cortical and hippocampal neurons isolated from the embryo of embryonic day 14-16 Sprague Dawley rats. This allows for the generation of neurospheres and long-term primary neuron culture as two independent platforms to conduct further studies. This process is extremely simple and cost-effective, as it minimizes several steps and reagents previously deemed essential for neuron culture. This is a robust protocol with minimal requirements that can be performed with achievable results and further used for a diversity of studies related to neuroscience.

Presented here is a protocol for the spontaneous generation of neurospheres enriched in neural progenitor cells from high density plated neurons. During the same experiment, when neurons are plated at a lower density, the protocol also results in prolonged primary rat neuron cultures.

从E14-E16斯普拉格·道利大鼠胚胎分离的混合原发希马和皮质神经元生成神经球

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have ...

小胶质细胞整合的视网膜类器官：研究视网膜疾病的模型

Assembling Retinal Organoids with Microglia

Due to the limited accessibility of the human retina, retinal organoids (ROs) are the best model for studying human retinal disease, which could reveal the mechanism of retinal development and the occurrence of retinal disease. Microglia (MG) are unique resident macrophages in the retina and central nervous system (CNS), serving crucial immunity functions. However, retinal organoids lack microglia since their differentiation origin is the yolk sac. The specific pathogenesis of microglia in these retinal diseases remains unclear; therefore, the establishment of a microglia-incorporated retinal organoid model turns out to be necessary. Here, we successfully constructed a co-cultured model of retinal organoids with microglia derived from human stem cells. In this article, we differentiated microglia and then co-cultured to retinal organoids in the early stage. As the incorporation of immune cells, this model provides an optimized platform for retinal disease modeling and drug screening to facilitate in-depth research on the pathogenesis and treatment of retinal and CNS-related diseases.

生成人类小胶质细胞以将它们与视网膜类器官相结合以改进疾病建模

Microglia are unique resident immune cells in the retina, playing crucial roles in various retinal degenerative diseases. Generating a co-culture model of retinal organoids with microglia can facilitate a better understanding of the pathogenesis and development progress of retinal diseases.

用小胶质细胞组装视网膜类器官

Due to the limited accessibility of the human retina, retinal organoids (ROs) are the best model for studying human retinal disease, which could reveal ...