Name: characterization and isolation of mouse primary microglia by density gradient centrifugation
Uploaded: 2018-02-16T13:30:00.000+00:00
Duration: 10 min 21 s
Description: Watch this Scientific Journal Video about Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation at JoVE.com

下列程序已获在莫纳什大学动物伦理学委员会批准。健康未经治疗的新生儿 C57Bl6/J P3-6 小鼠被用来产生代表性的结果。
1. 酶消化
注意: 在分离和培养主细胞时, 考虑不孕是很重要的。虽然确保环境尽可能的不育, 但最初的解剖和小鼠大脑的收获可以在层流流罩之外完成, 随后的所有步骤都在层流罩内进行。
<ol><li>使用无菌器械, 弄死小鼠颈椎脱位, 斩首动物, 用100% 乙醇冲洗。使用小的不育剪刀, 使小切口沿左右两侧的头部。在这个年龄, 皮肤很容易脱落。使用弯曲钳的尖端, 轻轻地滑向讲台上的组织, 以揭露头骨, 包括 Bregma。</li>
	<li>在椎管内的开口插入剪刀尖端, 并在耳道上进行侧向切口。沿矢状缝线切开 Bregma, 轻轻地指着剪刀的尖端, 以防止损伤大脑。在 Bregma 上插入剪刀尖端, 并沿冠状缝合在头部的左右两侧进行侧向切口。</li>
	<li>使用镊子, 轻轻剥去头骨暴露大脑。要做到这一点, 掌握侧切口暴露的颅骨边缘, 轻轻地将头骨拉到侧面。头骨应该很容易剥开。使用弯曲钳, 轻轻铲下大脑, 以消除它。</li>
	<li>将大脑放置在5毫升无菌容器中, 用冷洗介质 (低葡萄糖 Dulbecco 的改性鹰培养基 (DMEM) 补充1% 青霉素-链霉素), 大约5毫升介质每洗涤, 以去除血液。</li>
	<li>在层流气流罩, 放置5毫升容器的内容在一个小的培养皿 (35 毫米), 休息在冰上。使用无菌手术刀刀片或无菌剪刀, 取出小脑和嗅球。进行中线切割以分离两个半球。</li>
	<li>小心剥去脑膜层的细钳, 小心不要损伤皮质。将脑膜层识别为一层非常薄的细胞, 在大脑表面有红色的色调, 有明显的血管。保持脑部寒冷对于正确的脑膜摘除是必不可少的。如果脑膜层断裂, 继续剥去撕裂的碎片, 直到完全移除。</li>
	<li>将半球转移到一个新的小的培养皿 (在冰上), 并填充洗涤介质。用无菌手术刀刀片/剪刀把大脑切成小块。 注: 这些尺寸应为1毫米2 , 并且应注意不要将它们切成太小的部分, 因为这可能会降低产量。</li>
	<li>添加100µL 木瓜蛋白酶 (17 U/毫克股票) 和150µL DNase 我进入媒体和孵化30分钟在37°c</li>
	<li>消化后, triturate 组织使用 P1000 吸管。如果组织件太大而不能进入吸管, 考虑使用一对或无菌剪刀扩大吸管尖端。在此过程中, 注意不要将气泡引入媒体, 因为这可能会降低细胞的生存能力。</li>
</ol>2. 去除髓鞘碎片
<ol><li>在层流罩下使用无菌设备, 为每个大脑准备一个50毫升圆锥管, 100 µm 细胞过滤器。将培养皿中的内容倒入过滤器中, 将消化介质和脑片放在滤网上。通过使用不育的3毫升注射器的柱塞在研磨运动中通过大脑的片断, 直到没有更多的组织可见。消化后, 脑组织应该容易地瓦解。</li>
	<li>用洗涤介质连续清洗过滤器, 通过在整个过程中加满细胞滤网。这将洗涤通过过滤器中的任何细胞。 注意: 这应该做, 直到有约15毫升的管。这是为了确保过滤器得到充分的清洗, 并最大限度地增加所收集的细胞数量。</li>
	<li>
离心机单细胞悬浮在 500 x g 5 分钟在4摄氏度。在这段时间内, 准备密度梯度介质, 如所述。
	<ol>
<li>准备股票等渗 percoll (SIP), 这是密度梯度介质使用。通过增加9:1 密度梯度介质与10x 无菌汉克斯平衡盐溶液 (HBSS) 的比例来做到这一点。</li>
		<li>准备渐变为 30% sip 在 DMEM 和 70% sip 在 1x HBSS。例如, 准备10毫升 30% sip, 添加3毫升的 sip 到7毫升 DMEM。</li>
	</ol>
</li>
	<li>从锥形管中吸取上清, 在 DMEM 中并用重悬8毫升30% 口的细胞颗粒。把完整的体积转移到一个新鲜的15毫升圆锥管。</li>
	<li>衬 70% SIP 解决方案。要做到这一点, 填补一个转移吸管与 70% SIP, 并仔细推通过转移吸管到底部的锥形管。一旦尖端接近底部, 轻轻地推通过转移吸管的内容。 注意: 这样做缓慢, 以免扰乱30/70 界面。必须有一条清晰的线分隔两层。</li>
	<li>离心机的 SIP 层, 包括细胞, 在 650 x g 与刹车0和加速度 4, 在室温下25分钟。 注: 必须完成旋转与刹车关闭, 并允许离心机缓慢停止, 因为刹车的应用将扰乱界面, 大大减少了在 SIP 层之间的细胞收集。</li>
	<li>吸入管顶部的细胞碎片, 从顶部取出大约4毫升的介质。这将减轻在以下步骤中的单个核细胞的去除。使用 P1000 吸管, 小心地将吸管尖向下移向界面。将单个核细胞从30/70 密度梯度介质界面中分离出来。收集大约3毫升从多云接口和转移到一个新的15毫升圆锥管。在此之后, 用9毫升的 HBSS 稀释混合物, 以帮助去除密度梯度介质。</li>
	<li>离心稀释密度梯度介质界面, 包含单个核细胞 500 x g 5 分钟, 吸出上清和并用重悬1毫升生长培养基 (DMEM 补充10% 胎牛血清和1% 抗生素)。接着, 用台盼蓝染色悬浮细胞, 用 haemocytometer 进行细胞计数。</li>
</ol>3. 磁性活化细胞分类
注意: 这些步骤是从制造商协议中修改的。
<ol><li>将所收集的单个核细胞以4°c 的 300 x g 为10分钟, 以去除生长介质, 因为这会干扰磁隔离。使用从步骤2.8 获得的相同单元格计数, 并用重悬在 PBS (Ca ++ 和Mg ++ 免费) 中的 1 x10 8 有核细胞/mL, 其中包含2% 个血清和1毫米 EDTA, 在体积范围内为 0.1-2.5 毫升。</li>
	<li>从上一步到5毫升 (12 x 75 毫米) 聚苯乙烯圆底管, 在 PBS 中添加完整的有核细胞。添加50µL CD11b PE 标签试剂每1毫升的样品。在室温下孵化15分钟免受光照。</li>
	<li>添加70µL 的选择鸡尾酒 (CD11b 单克隆抗体的组合) 每1毫升的样品。在室温下孵化15分钟免受光照。</li>
	<li>通过吹打混合磁性粒子5倍以上。将50µL/毫升添加到样品中。在室温下孵化10分钟免受光照。 注意: 这些微粒然后形成一个 tetrameric 复合物与抗体并且将被连接到磁性专栏。</li>
	<li>如果细胞混合物的总体积小于2.5 毫升, 顶部由 PBS (Ca++和 Mg++免费) 组成, 含有2% 的血清和1毫米 EDTA, 并通过轻轻吹打2-3 次混合。把管子 (没有盖子) 放进磁铁里, 在室温下孵育5分钟。</li>
	<li>在一个连续的运动中, 完全反转含有 2-3 s 管的磁铁, 倒出上清。将磁铁返回到直立位置, 然后从磁铁中取出管。准备从列中清洗剩余的单元格。 注: 上清中含有无标号和不需要的细胞, 通过反转管, 同时仍在磁铁中去除。该管包含附加的 Cd11b+单元格。</li>
	<li>通过重复步骤3.5 和3.6 两次, 来洗涤细胞。如果样品大于1毫升, 建议进一步重复步骤3.5 和3.6。</li>
	<li>并用重悬所需的生长培养基中的细胞。同时冲洗收集容器的侧面 (e. g. 15 毫升管) 从管的两侧收集细胞, 并使产量最大化。</li>
</ol>4. 小胶质细胞纯度的验证
注: 从3升 (n = 5 动物总数) 中分离出的主要小胶质细胞通过荧光活化单元分类 (资产管制仪) 进行验证, 以确定代表结果的纯度。
<ol><li>T-25 瓶中的培养细胞, 其生长培养基中含有10% 的血清, 每瓶的密度为 1-2 x 106细胞。</li>
	<li>用 PBS 3x 将 T-25 烧瓶中的细胞洗净5分钟, 以去除可能干扰 trypsinization 的任何剩余生长介质。</li>
	<li>加入2毫升0.25% 胰蛋白酶, 从烧瓶中分离出细胞。确保胰蛋白酶的体积足以覆盖整个瓶子的表面。下面的烧瓶温和旋转, 以确保胰蛋白酶均匀覆盖, 孵育5分钟在37摄氏度。</li>
	<li>通过在烧瓶中加入2毫升的生长培养基, 其中含有10% 的血清, 从而使 trypsinization 过程淬火。</li>
	<li>收集含有 trypsinized 细胞和离心机在 500 x g 5 分钟在4摄氏度收集细胞的上清液。</li>
	<li>移除含有生长培养基和胰蛋白酶的上清液, 并在 500 ul 的并用重悬中对颗粒进行清除。使用台盼蓝进行细胞计数。</li>
	<li>离心机在 500 x g 5 分钟在4摄氏度。在每 5 x 106单元格中添加 50 ul 操作器缓冲区 + 1 ul FcR 拦截器, 执行 Fc 受体块。孵育15分钟, 在4摄氏度。 注意: fc 受体阻滞是染色过程中的一个关键步骤, 以防止免疫球蛋白的非特异性结合到 Fc 受体细胞中。这种封锁不影响其他抗体的下游约束力。</li>
	<li>通过稀释 500 ul 流化器缓冲区来终止阻塞过程。在此之后, 离心机的混合物包含细胞在 500 x g 5 分钟在4摄氏度。</li>
	<li>并用重悬在新鲜的 500 ul 的资产管制器缓冲区。</li>
	<li>放置大多数单元格 (例如, 如果将 resuspending 500 ul 中的单元格, 使用 400 ul) 放入取样管中。将剩余的电池分布在控制管中, 每个控制管最多可达100个 ul (例如, 6 个控制管, 将 16.67 ul 放入每个控制管中, 并以 83.33 ul 的流控器缓冲区为顶部)。使组 (控制管以粗体) 为无瑕, 单染色 (CD45, CD11b), 单活/死染色, FMOs 和样品管。</li>
	<li>通过离心在 500 x g 5 分钟, 在4摄氏度将所有管子中的细胞颗粒状。</li>
	<li>每 5 x 106单元格中, 用 1 ul 抗体对管中的细胞进行染色, 每 100 ul 的检测器缓冲区。孵育20分钟, 在4摄氏度。 注: 如果使用少于 2 x 106单元格, 请使用 50 ul 抗体鸡尾酒。</li>
	<li>用 500 ul 的流器缓冲器清洗细胞。离心细胞在 500 x g 5 分钟在4摄氏度。</li>
	<li>并用重悬 300 ul 的传感器缓冲区中的单元格。 注: 如果单元格计数小于 2 x 106, 则使用 100 ul。</li>
	<li>使用流式细胞仪分析, 量化 CD45低和阳性 CD11b 染色的细胞。这个百分比对应于被隔绝的主要小胶质细胞。</li>
</ol>5. 原发性小胶质细胞的免疫组化染色
<ol><li>将96井板中的细胞以 1 x 105细胞的密度与生长培养基结合, 并在一夜之间孵化。</li>
	<li>取出上清液, 用 PBS 清洗3次。</li>
	<li>将4% 多聚甲醛的单元格固定在 PBS 中15分钟. 用 P1000 吸管取出粉煤灰, 然后用 pbs + 0.1% 海卫 x 清洗3次。</li>
	<li>室温下以3% 牛血清白蛋白为1小时的非特异结合块。</li>
	<li>孵育与兔子 Iba-1 (1:100) 为24小时在4°c。接着, 取出主要抗体混合物, 用 PBS 清洗3次。</li>
	<li>二次抗兔 GFP 共轭 (1:200) 在室温下孵育1小时。接着, 取出二次抗体混合物, 用 PBS 清洗3次。</li>
	<li>安装带有荧光安装介质的幻灯片, 并使用荧光显微镜捕捉图像。</li>
</ol>6. 用 pHrodo 测定小胶质细胞的数量
注: pHrodo 化验可以识别培养细胞中吞噬功能的水平。通过吞吸收, 内部化进入更酸性的环境, 增加了 bioparticle 共轭物的荧光水平。然后, 可以通过外地资产管制仪量化荧光水平。以下步骤由制造商协议修改。
<ol><li>一夜之间在生长培养基中培养 T-25 烧瓶中的细胞, 每瓶的密度为 1-2 x 106细胞。用 PBS 将 T-25 烧瓶中的细胞洗净3次, 5 分钟。</li>
	<li>使用2毫升0.25% 胰蛋白酶分离细胞。孵育5分钟, 在37摄氏度。</li>
	<li>通过在烧瓶中加入2毫升的生长培养基, 其中含有10% 的血清, 从而使 trypsinisation 过程淬火。</li>
	<li>离心细胞悬浮在 500 x g 为5分钟在4°c 到颗粒细胞。</li>
	<li>移除上清和并用重悬在10 6 细胞/mL 培养基中含有主要小胶质的小球。 注: 另一种情况是, 96 井板中的平板细胞至少在一天前, 并在进行化验的当天以 1 x 105可行细胞为目标。</li>
	<li>板单元成96井板在 1 x 105细胞/好, 100 ul 每井。板控制和实验井三个, 并留下一个空井每积极控制的无细胞控制背景减法。</li>
	<li>添加 100 ul 的增长介质到井留空为无细胞背景减法。 注意: 与任何其他体外检测一样, 适当的控制对于读数的准确性至关重要。除了无细胞控制 (背景阅读), 还包括负控制井 (pHrodo 共轭添加, 但放置在冰上)。</li>
	<li>盖板和孵化1小时在一个孵化器与 5% CO2在37摄氏度。</li>
	<li>通过添加兴奋剂和治疗来制备实验井。将车辆控制 (仅增长媒体) 添加到未经处理的油井。 注: 脂多糖1µg/毫升 [兴奋剂] 和人羊膜上皮细胞 (hAEC) 条件的媒体 [治疗] 被用来产生代表性的结果。</li>
	<li>解冻一小瓶共轭染料, 加入2毫升吸收缓冲和简要涡。将管的内容转化为干净的玻璃管和油脂实验5分钟, 以确保颗粒均匀分散。</li>
	<li>从微板块井中吸取培养基, 用 100 ul 的染料悬浮液快速替换。覆盖和孵化在37°c 1 小时。 注: 请记住, 也要弄脏控制管。</li>
	<li>应用流式细胞仪分析, 量化细胞染色阳性的共轭 pHrodo 珠, 目前作为一个百分比 (父母) 的积极吞噬小胶质。</li>
</ol>7. 炎症性侮辱后小胶质细胞凋亡的量化
<ol><li>重复步骤1-7 从 "验证小胶质细胞纯度" 一节。</li>
	<li>放置大多数单元格 (例如, 如果您将 500 ul 的悬浮中的单元格, 使用 400 ul) 放入取样管中。将剩余的电池分布在控制管中, 每个控制管最高可达 100 ul (例如6 控制管, 将 16.67 ul 放入每个控制管中, 并以 83.33 ul 的流控器缓冲区为顶部)。组 (控制管粗体): 无瑕, 单污渍 (AnnexinV, 碘碘化), 单活/死染色, FMOs, 样品管。</li>
	<li>通过离心在 500 x g 5 分钟内将所有管子中的细胞颗粒。</li>
	<li>孵育与 1 ul antibody/100 ul buffer/5 x 106单元格为20分钟在4°c。 注: 如果少于 2 x 106单元格, 请使用 50 ul。</li>
	<li>在 500 x g 处添加 500 ul 的流道缓冲液和离心机以5分钟冲洗细胞。</li>
	<li>并用重悬在 300 ul 的内流器缓冲器中的细胞颗粒。 注: 如果单元格计数小于 2 x 106, 则为 100 ul。</li>
	<li>量化每个象限内的细胞 (Q1: 坏死, Q2: 晚期凋亡, Q3: 可行, Q4: 早期凋亡)。</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Debris+clearance+by+microglia:+an+essential+link+between+degeneration+and+regeneration.">Debris clearance by microglia: an essential link between degeneration and regeneration.</a> Brain. 132 (2), 288-295 (2009).</li><li>Veronique, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M2+microglia+and+macrophages+drive+oligodendrocyte+differentiation+during+CNS+remyelination.">M2 microglia and macrophages drive oligodendrocyte differentiation during CNS remyelination.</a> Nature Neuroscience. 16 (9), 1211 (2013).</li><li>Hu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia/macrophage+polarization+dynamics+reveal+novel+mechanism+of+injury+expansion+after+focal+cerebral+ischemia.">Microglia/macrophage polarization dynamics reveal novel mechanism of injury expansion after focal cerebral ischemia.</a> Stroke. 43 (11), 3063 (2012).</li><li>Kigerl, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+two+distinct+macrophage+subsets+with+divergent+effects+causing+either+neurotoxicity+or+regeneration+in+the+injured+mouse+spinal+cord.">Identification of two distinct macrophage subsets with divergent effects causing either neurotoxicity or regeneration in the injured mouse spinal cord.</a> The Journal of Neuroscience : the official journal of the Society for Neuroscience. 29 (43), 13435 (2009).</li><li>Suzuki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+&#945;7+nicotinic+acetylcholine+receptors+drive+a+phospholipase+C/IP3+pathway+and+modulate+the+cell+activation+toward+a+neuroprotective+role.">Microglial &#945;7 nicotinic acetylcholine receptors drive a phospholipase C/IP3 pathway and modulate the cell activation toward a neuroprotective role.</a> Journal of neuroscience research. 83, (2006).</li><li>Bedi, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravenous+multipotent+adult+progenitor+cell+therapy+attenuates+activated+microglial/macrophage+response+and+improves+spatial+learning+after+traumatic+brain+injury.">Intravenous multipotent adult progenitor cell therapy attenuates activated microglial/macrophage response and improves spatial learning after traumatic brain injury.</a> Stem cells translational medicine. 2, (2013).</li><li>Tran, T. A., McCoy, M. K., Sporn, M. B., Tansey, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+synthetic+triterpenoid+CDDO-methyl+ester+modulates+microglial+activities,+inhibits+TNF+production,+and+provides+dopaminergic+neuroprotection.">The synthetic triterpenoid CDDO-methyl ester modulates microglial activities, inhibits TNF production, and provides dopaminergic neuroprotection.</a> Journal of neuroinflammation. 5, 14 (2008).</li><li>Gr&#252;tzkau, A., Radbruch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+but+mighty:+How+the+MACS&#174;-technology+based+on+nanosized+superparamagnetic+particles+has+helped+to+analyze+the+immune+system+within+the+last+20+years.">Small but mighty: How the MACS&#174;-technology based on nanosized superparamagnetic particles has helped to analyze the immune system within the last 20 years.</a> Cytometry Part A. 77A (7), 643-647 (2010).</li><li>Nikodemova, M., Watters, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+isolation+of+live+microglia+with+preserved+phenotypes+from+adult+mouse+brain.">Efficient isolation of live microglia with preserved phenotypes from adult mouse brain.</a> Journal of Neuroinflammation. 9, 147-147 (2012).</li><li>Holt, L. M., Olsen, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+Applications+of+Magnetic+Cell+Sorting+to+Analyze+Cell-Type+Specific+Gene+and+Protein+Expression+in+the+Central+Nervous+System.">Novel Applications of Magnetic Cell Sorting to Analyze Cell-Type Specific Gene and Protein Expression in the Central Nervous System.</a> PLOS ONE. 11 (2), e0150290 (2016).</li><li>Leaw, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+amnion+epithelial+cells+rescue+cell+death+via+immunomodulation+of+microglia+in+a+mouse+model+of+perinatal+brain+injury.">Human amnion epithelial cells rescue cell death via immunomodulation of microglia in a mouse model of perinatal brain injury.</a> Stem cell research &amp; therapy. 8 (1), 46 (2017).</li><li>Moujalled, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TDP-43+mutations+causing+amyotrophic+lateral+sclerosis+are+associated+with+altered+expression+of+RNA-binding+protein+hnRNP+K+and+affect+the+Nrf2+antioxidant+pathway.">TDP-43 mutations causing amyotrophic lateral sclerosis are associated with altered expression of RNA-binding protein hnRNP K and affect the Nrf2 antioxidant pathway.</a> Human Molecular Genetics. 26 (9), 1732-1746 (2017).</li></ol>

作者没有什么可透露的。

小胶质细胞具有支持和抗炎的能力, 通过环境刺激改变。先前的研究表明, 小胶质细胞活化的调节可以赋予神经保护作用。他们为神经元提供保护和修复损伤的能力需要更多的研究来进一步了解这些复杂细胞的现状。因此, 高纯度原发性小胶质细胞的分离是一项重要而有用的技术。这是一个相对较快的方法, 获得高纯度的主要小胶质细胞为体外实验在2天内准备好。
文献?...

围产期期间从炎症、缺氧缺血性和出血中获得的损伤可以有一系列的长期后遗症。围产期脑损伤的复杂病理生理学理论涉及炎症和缺血与随后的神经和轴突死亡1。先天免疫应答在导致伤害的一连串事件中起重要作用2。
小胶质细胞, 中枢神经系统 (CNS) 内的居民免疫单元, 是伤害3的第一个应答者。小胶质细胞是具有保护或毒性能力的塑料电池类型, 依赖于环境4。它们参与趋化、吞噬、抗原表达和细胞因子的产生和活性氧种类的4,5。衰老小胶质细胞不断地对环境进行调查, 并由异物或有害物质的存在4激活。激活会导致一种支持炎症的响应, 在 CNS 保护4中至关重要。这些 M1 的 "亲炎" 表型小胶质细胞主要参与抗原表示和病原体的死亡4。尽管炎症反应在神经保护中起着关键作用, 但不受控制或长时间的炎症可能有害, 导致神经元损伤4。然而, 当暴露在某些环境刺激, 小胶质细胞可以表现出一种抗炎的表型。这些支持修复的 M2 小胶质细胞在伤口愈合和修复过程中具有关键作用6, 释放出一系列 downregulate 炎症、增加吞噬功能和促进修复4, 7. 小胶质细胞的作用多种多样, 包括在髓鞘形成中驱动少突胶质分化 8, 在中风模型的氧和葡萄糖耗尽期间保护神经元9并促进突起在脊髓损伤模型10。
对这些胶质细胞的研究是理解和操作 neuroinflammation 反应的一个重要方面。所述的协议允许进一步研究小胶质细胞调制在 neuroinflammatory 疾病中的治疗潜力。
胶质激活对神经保护角色的调制被观察到了一系列条件11,12,13。因此, 提高当前的理解和进一步研究胶质激活的调制是至关重要的, 需要使用包括体外和体内在内的各种模型。体外研究是一种重要的工具, 因为它们具有更高的效率、更低的成本和调查孤立细胞群体的能力。
文献中描述了一系列的协议, 用于从小鼠脑中分离小胶质细胞, 从而有效地生产出高产样品, 具有良好的生存能力和高纯度。常见的小胶质细胞的分离方法是通过磁分离和长时间晃动的混合胶质细胞培养。通过个人经验发现, 有很大程度的细胞碎片阻碍了磁柱。因此, 使用了下面的协议, 其中包括初始密度梯度离心步骤后, CD11b 磁分离。下面描述的协议已经过优化, 以产生足够数量的高度纯样本。它是有利的由于它的高纯度和短的时间周期-你可以在2天内进行化验, 而无需培养2-3 周。该协议有可能被改编为小鼠星形胶质细胞的分离。

<table><tbody><tr><td>DMEM, low glucose, pyruvate</td><td>Gibco</td><td>11885084</td><td></td></tr><tr><td>Antibiotic-Antimycotic (100X)</td><td>Gibco</td><td>15240062</td><td></td></tr><tr><td>DNaseI grade II from bovine pancreas</td><td>Sigma-Aldrich</td><td>10104159001</td><td></td></tr><tr><td>Papain from papaya latex, buffered aqeuous solution</td><td>Sigma-Aldrich</td><td>P3125-100mg</td><td></td></tr><tr><td>Fetal Bovine Serum, qualified, heat inactivated</td><td>Gibco</td><td>16140071</td><td></td></tr><tr><td>Percoll</td><td>GE Healthcare</td><td>17-0891-01</td><td></td></tr><tr><td>Hank&#039;s Balanced Salt Solution (1X)</td><td>Gibco</td><td>14175-103</td><td></td></tr><tr><td>Hank&#039;s Balanced Salt Solution (10X)</td><td>Gibco</td><td>14185052</td><td></td></tr><tr><td>EasySep Mouse CD11b Positive Selection Kit</td><td>StemCell Technologies</td><td>18770</td><td>EasySep magnet variant</td></tr><tr><td>EasySep magnet</td><td>StemCell Technologies</td><td>18000</td><td></td></tr><tr><td>EasySep Buffer</td><td>StemCell Technologies</td><td>20144</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate buffered saline</td><td>Gibco</td><td>14040182</td><td></td></tr><tr><td>Trypsin (2.5%) (10X)</td><td>Gibco</td><td>15090-046</td><td></td></tr><tr><td>Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block&amp;trade;)</td><td>BD Biosciences</td><td>553141</td><td></td></tr><tr><td>Falcon 5mL Round Bottom High Clarity PP Test Tube, with Snap Cap, Sterile</td><td>Corning</td><td>352063</td><td></td></tr><tr><td>175cm&amp;sup2; Angled Neck Cell Culture Flask with Vent Cap</td><td>Corning</td><td>431080</td><td></td></tr><tr><td>Lipopolysaccharides from Escherichia coli O127:B8</td><td>Sigma-Aldrich</td><td>L5024</td><td></td></tr><tr><td>96 Well TC-Treated Microplates size 96 wells, clear, polystyrene, round bottom</td><td>Corning</td><td>CLS3799</td><td></td></tr><tr><td>Paraformaldehyde (powder, 95%)</td><td>Sigma-Aldrich</td><td>158127</td><td></td></tr><tr><td>Triton-X</td><td>Sigma-Aldrich</td><td>X100</td><td></td></tr><tr><td>Rabbit Anti-Iba1</td><td>Wako</td><td>01919741&amp;nbsp;</td><td></td></tr><tr><td>Goat Anti-Rabbit IgG H&amp;amp;L (Alexa Fluor 488)</td><td>Abcam</td><td>ab150077</td><td></td></tr><tr><td>&lt;strong&gt;FACS Antibodies&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td></td></tr><tr><td>V450,Rat,Anti-Mouse,CD45,30-F11,RUO</td><td>BD Biosciences</td><td>560501</td><td></td></tr><tr><td>PerCP-Cy5.5 CD11b&amp;nbsp;</td><td>eBiosciences</td><td>45-0112-82</td><td></td></tr><tr><td>ZombieNIR</td><td>Biolegend</td><td>423105</td><td></td></tr><tr><td>pHrodo Red E. coli BioParticles Conjugate</td><td>Thermo Fisher Scientific</td><td>P35361</td><td></td></tr><tr><td>Annexin.V_FITC</td><td>Miltenyi Biotech</td><td>130-093-060</td><td></td></tr><tr><td>Propodium Iodide solution</td><td>Miltenyi Biotech</td><td>130-093-233</td><td></td></tr></tbody></table>

characterization and isolation of mouse primary microglia by density gradient centrifugation

小胶质细胞, 是大脑中的驻留免疫单元, 是中枢神经系统炎症或损伤的第一反应者。最近的研究发现小胶质细胞是动态的, 能够同时承担炎症和抗炎的表型。两种 M1 (亲炎症) 和 M2 (支持修复) 表型在诸如围产期脑损伤等 neuroinflammatory 条件下起着重要作用, 并表现出不同的功能, 以应对某些环境刺激。胶质激活的调节已被注意到赋予神经保护, 因此提示小胶质细胞可能有治疗潜力的脑损伤。然而, 需要更多的研究, 以更好地了解小胶质细胞在疾病中的作用, 这项议定书有利于这一点。下面描述的协议结合了密度梯度离心过程, 以减少细胞碎片, 与磁性分离, 产生一个高度纯净的主要胶质细胞样本, 可用于体外实验, 没有需要2-3 周的培养。此外, 这些表征步骤产生了关于小胶质细胞的强健的功能数据, 帮助研究, 以更好地了解这两种干细胞的极化和启动, 这在再生医学领域具有很强的意义。

使用此处概述的方法, 可以隔离小胶质细胞的纯种群, 并可以使用体外和资产管制分析来进行表征。首先, 多达18只动物可以使用每一个剔除, 预期产量约 45万-60万胶质细胞。首先确认分离细胞的纯度是至关重要的, 这样做是通过对两个标记 CD45 和 CD11b 的染色来进行的. 胶质细胞的鉴定可以证明是麻烦的, 因为小胶质细胞表达的许多标记也表达了巨噬细胞, 并使用这两个...

Watch this Scientific Journal Video about Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation at JoVE.com

Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation

密度梯度离心法分离小鼠原发胶质细胞的研究

本文提出了一种从小鼠脑中分离原发胶质细胞的方法。这种技术有助于促进目前对神经系统状况的理解。将密度梯度离心和磁分离相结合, 产生高纯样品的充分屈服。此外, 我们概述了小胶质细胞的表征步骤。

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

characterization-isolation-mouse-primary-microglia-density-gradient

Research

JoVE Journal

Neuroscience

19.2K 次观看.  Hudson Institute of Medical Research. 本文提出了一种从小鼠脑中分离原发胶质细胞的方法。这种技术有助于促进目前对神经系统状况的理解。将密度梯度离心和磁分离相结合, 产生高纯样品的充分屈服。此外, 我们概述了小胶质细胞的表征步骤。

视频: Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation

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 ...

科研

免疫与感染

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. ...

神经科学

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

Microglia represent 5 - 10% of all central nervous system (CNS) cells and are increasingly drawing attention due to their contributions during development, homeostasis, and disease. Although macrophages have been studied in detail for decades, specialized features of microglia, the tissue-resident macrophages of the CNS, have remained largely mysterious, in part due to limitations in the ability to recapitulate mature microglial properties in culture. Here, we illustrate a straightforward procedure for the rapid isolation of pure microglia from the mature rodent brain. We also describe serum-free culture conditions that support high levels of microglial viability over time. Microglia cultured under these defined-medium conditions exhibit elaborate ramified processes and dynamic surveillance behavior. We illustrate some effects of serum exposure on cultured microglia and discuss how these serum-free cultures compare to both serum-exposed cultures as well as microglia in vivo.

Efforts to understand microglial function in detail have been hindered by the lack of microglial culture models that recapitulate the properties of mature in vivo microglia. This protocol describes an isolation and culture approach designed to maintain robust survival of highly ramified mature rat microglia under defined-medium conditions.

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 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.

从一个成年小鼠脑半球分离区域特异性微胶质，进行深度单细胞RNA测序

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

Primary Microglia Isolation from Postnatal Mouse Brains

Microglia are the mononuclear phagocytes in the central nervous system (CNS), which play key roles in maintaining homeostasis and regulating the inflammatory process in the CNS. To study the microglial biology in vitro, primary microglia show great advantages compared to immortalized microglial cell lines. However, microglia isolation from the postnatal mouse brain is relatively less efficient and time-consuming. In this protocol, we provide a quick and easy-to-follow method to isolate primary microglia from the neonatal mouse brain. The overall steps of this protocol include brain dissection, primary brain cell culture, and microglia isolation. Using this approach, researchers can obtain primary microglia with high purity. In addition, the harvested primary microglia were able to respond to the lipopolysaccharides challenge, indicating they retained their immune function. Collectively, we developed a simplified approach to efficiently isolate primary microglia with high purity, which facilitates a wide range of microglial biology investigations&#160;in vitro.

<meta charset="utf8"></head><body>从出生后小鼠大脑中分离原代小胶质细胞

Primary cell culture is one of the primarily used approaches for studying microglial biology in vitro. Here, we developed a method for simple and rapid microglia isolation from the mouse postnatal day 1 (P1) to P4.

从出生后小鼠大脑中分离原代小胶质细胞

Microglia are the mononuclear phagocytes in the central nervous system (CNS), which play key roles in maintaining homeostasis and regulating the ...

Isolation of Mouse Primary Microglia by Magnetic-Activated Cell Sorting in Animal Models of Demyelination

Microglia, the resident innate immune cells in the brain, are the primary responders to inflammation or injury in the central nervous system (CNS). Microglia can be divided into resting state and activated state and can rapidly change state in response to the microenvironment of the brain. Microglia will be activated under different pathological conditions and exhibit different phenotypes. In addition, there are many different subgroups of activated microglia and great heterogeneity between different subgroups. The heterogeneity mainly depends on the molecular specificity of microglia. Studies have revealed that microglia will be activated and play an important role in the pathological process of inflammatory demyelination. To better understand the characteristics of microglia in inflammatory demyelinating diseases such as multiple sclerosis and neuromyelitis optica spectrum disorder, we propose a perilesional primary microglial sorting protocol. This protocol utilizes columnar magnetic-activated cell sorting (MACS) to obtain highly purified primary microglia and preserve the molecular characteristics of microglia to investigate the potential effects of microglia in inflammatory demyelinating diseases.

Here, we present a protocol to isolate and purify primary microglia in animal models of demyelinating diseases, utilizing columnar magnetic-activated cell sorting.

通过磁活化细胞分选在脱髓鞘动物模型中分离小鼠原代小胶质细胞

Microglia, the resident innate immune cells in the brain, are the primary responders to inflammation or injury in the central nervous system (CNS). ...

使用 MACS 从小鼠中分离下丘脑小胶质细胞进行体外测定

Isolation of Hypothalamic Microglia from Freshly Perfused Adult Mouse Brain by Magnetic-Activated Cell Sorting

Microglia, as the resident macrophages of the brain, are essential for maintaining brain homeostasis. They shape neuronal circuits during development, survey their environment for debris or dead cells, as well as respond to infection and injury in the brain, among many other functions. However, their important role in neurodevelopment and synaptic plasticity and pathophysiology has not been fully defined, highlighting the need for further investigation. To gain a more comprehensive understanding of the role of microglia in these processes, we need to isolate microglia and characterize them genetically, metabolically, and functionally. However, the isolation of microglia from adult mice, especially from small brain structures, is challenging as they represent a small percentage of the total brain cells, and the yield of isolated microglia is often too low. Here, the magnetic isolation of microglia using CD11b+ microbeads allows us to sort microglial cells from the hypothalamus of a freshly perfused adult mouse brain. The current method allows us to achieve relatively high purity and yield in a short period while maintaining cell viability.

作者聚焦:研究营养对小鼠脑功能和代谢紊乱的影响 

We describe a protocol for isolating microglial cells from the mouse hypothalamus (or equivalent small brain structures) using magnetically activated cell sorting (MACS), in a relatively short time. The MACS-sorted hypothalamic microglia can be used for ex vivo analysis and can be plated to perform in vitro assays.

通过磁激活细胞分选从新鲜灌注的成年小鼠脑中分离下丘脑小胶质细胞  

Microglia, as the resident macrophages of the brain, are essential for maintaining brain homeostasis. They shape neuronal circuits during development, ...

密度梯度离心法分离小鼠原发胶质细胞的研究

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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

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从一个成年小鼠脑半球分离区域特异性微胶质，进行深度单细胞RNA测序

从出生后小鼠大脑中分离原代小胶质细胞

从出生后小鼠大脑中分离原代小胶质细胞

通过磁活化细胞分选在脱髓鞘动物模型中分离小鼠原代小胶质细胞

通过磁激活细胞分选从新鲜灌注的成年小鼠脑中分离下丘脑小胶质细胞

通过磁激活细胞分选从新鲜灌注的成年小鼠脑中分离下丘脑小胶质细胞

通过磁激活细胞分选从新鲜灌注的成年小鼠脑中分离下丘脑小胶质细胞