该议定书得到了大韩民国科学、信息和通信技术和未来规划部赠款 NRF-2015R1A2A1A01003410 的支持。

所有涉及动物的实验都得到了 Ajou 大学医学院动物保育和使用委员会的批准。
1. 游离成人背根神经节神经元的培养制备
<ol><li>在建立培养之前, 先用聚 d-赖氨酸和层粘连蛋白对6井板进行预涂。在37°c 孵化一个6井板, 0.01% 聚 d-赖氨酸, 在2小时或在4°c 上过夜。然后, 用蒸馏水洗两次盘子。</li>
	<li>在室温下, 用层粘连蛋白溶液在3µg/毫升浓度下孵育板材, 然后用蒸馏水冲洗两次盘子。至少在室温下将板材干燥1小时。</li>
	<li>弄死与压缩 co2气瓶相连的透明 co2腔中的鼠标。用手术刀片切开椎柱上的皮肤, 并通过双边解剖旁的肌肉以暴露椎体的骨骼。使用狭窄的外科钳, 在 DRGs 完全暴露之前, 小心切除椎体骨骼。 注: 成年 DRGs 神经元由成年 C57BL6 雄性小鼠, 年龄介于8周至12周之间。</li>
	<li>使用虹膜剪刀和细尖钳在解剖显微镜下, 从 S1 一直到 C1 水平 DRGs。尽量从 DRGs 中切下根部, 以尽量减少雪旺细胞或成纤维细胞的污染。</li>
	<li>将解剖过的 DRGs 保存在60毫米培养皿中, 其中5毫升冷 Dulbecco 的修饰鹰培养基 (DMEM) 在冰上, 直到所有的 DRGs 都被收集。 注意: 尽快解剖 DRGs 是至关重要的。所有 DRGs 从一只老鼠的收集不应超过30分钟30分钟后, 安乐死, 停止收集 DRGs, 并继续下一步, 即使仍然有剩余的 DRGs。</li>
	<li>使用蓝色吸管尖端 (1000 µL) 将 DRGs 转移到1.5 毫升离心管, 并切断末端。使用微型离心机, 在快速旋转几秒钟后, 取出 DMEM。添加1毫升的 DMEM 含有 125 U/毫升类型的西胶原酶和孵化管90分钟以温和的旋转 (35-40 rpm) 使用扭振器在37°c 孵化器。用胶带固定在振动筛地板上的管子。</li>
	<li>放弃含胶原酶的 DMEM, 加入1毫升新鲜 DMEM。等待, 直到漂浮的 DRGs 完全下沉到底部, 然后删除 DMEM 与组织碎片。重复这个洗涤步骤至少六次。 注意: 不要试图完全丢弃上清。小心不要删除任何 DRGs 与上清 DMEM。</li>
	<li>使用蓝色吸管尖端 (1000 µL) 将 DRGs 转移到15毫升圆锥管, 并切断末端。然后用蓝尖 (1000 µL) 轻轻地向上和向下吸15次, 使均匀的细胞悬浮。避免制造气泡, 尽量不要用吸管尖触碰锥形管的底部。</li>
	<li>离心管在 239 x g 3 分钟, 并小心地丢弃上清上漂浮的碎片。添加1毫升的 Neurobasal 培养基, 辅以 B27 (2.0% 伏/v), 并并用重悬细胞颗粒轻轻吹打向上和向下5到10倍。</li>
	<li>通过 70-µm 细胞过滤器覆盖在50毫升圆锥管顶部的细胞悬浮。等待2分钟, 然后用吸管控制器将 Neurobasal/B-27 介质倒入过滤器上。</li>
	<li>板所有收集的背根神经节神经元 (总共约 2 x 106背节神经元从一只老鼠) 到两个井的6井板。一只成年小鼠的背根神经节在6井板上覆盖两口井。然后将该板块放置在37°c 的 co2孵化器中, 直到巨噬细胞共培养。</li>
</ol>2. P 原发性腹腔巨噬细胞的共培养
注: 建立离型背根神经节神经元初始电镀后4小时的共培养
<ol><li>原发腹腔巨噬细胞由成年 C57BL6 雄性小鼠在8周至12周龄之间制备。弄死在 CO2腔中的动物。细致切开腹部皮肤, 暴露腹膜, 避免切开腹膜, 防止灌洗液渗漏。</li>
	<li>用一根22G 针的注射器刺穿腹膜, 将10毫升的冰冻磷酸盐缓冲 salin (PBS) 注入腹腔。轻轻按摩腹膜1-2 分钟。然后, 拔出针头, 通过穿刺部位挤出 PBS, 在50毫升锥形管中收集灌洗液。 注: 使用前, 把注射器放在冰上, 以保持 PBS 的寒冷。</li>
	<li>离心冲洗液在 239 x g 10 分钟在4°c, 以颗粒的细胞成分。并用重悬3毫升的红细胞 (红细胞) 裂解缓冲液在室温下为3分钟。然后离心细胞悬浮在 239 x g 10 分钟在4°c。并用重悬颗粒与3毫升 PBS 和离心机细胞悬浮再次删除任何剩余的红细胞溶解缓冲。 注意: 确保红细胞溶解缓冲液完全清除。否则剩余的红细胞溶解缓冲液可能对培养的巨噬细胞有毒。</li>
	<li>并用重悬1毫升 Neurobasal/B-27 培养基中的颗粒细胞。所有收集到的巨噬细胞的一半 (总共 3 x 106到 6 x 106单元格) 在单元格区域性插入与有效面积 4.2 cm2, 它被放置在分离的背根的井的顶部。 注: 在共培养期, 巨噬细胞生长在 Neurobasal/B-27 神经元培养基中。在这种情况下证实了巨噬细胞的充分存活。</li>
</ol>3. 治疗 Db 营和收集巨噬细胞 CM
注: 在神经元-巨噬细胞联合培养后开始4小时的 db 营治疗。
<ol><li>添加2µL 100 µM db 阵营解决方案的神经元-巨噬细胞共培养。为控制实验添加相同体积的 PBS。</li>
	<li>24小时后, 用1毫升的巨噬细胞培养培养基在同一6井板中填充空井。用巨噬细胞培养培养基将神经元-巨噬细胞共培养物中的胞培养物转移到空井。保持细胞在相同的条件下72小时, 而不改变培养基。 注: 在 cm 收集过程中, 我们只添加1毫升的巨噬细胞培养基, 以使浓缩厘米。在 CM 收集, 如果需要, 我们增加了更多的完全覆盖在插入的巨噬细胞。</li>
	<li>在 72 h 以后, 离心机巨噬细胞 CM 在 239 x g 为5分钟去除细胞组分。通过0.2 µm 过滤器将上清液移除其余的细胞碎片。将收集的 CM 保存在-70 摄氏度, 直到使用。</li>
</ol>4. 用收集到的 CM 突起生长试验
<ol><li>在建立一个单独的成人背根神经节神经元培养之前, 在1.1 和1.2 中所描述的相同的过程中, 预先涂上8井室滑块。</li>
	<li>用从1.3 到1.10 的相同方法获得 Neurobasal 培养基中游离的成人背根神经节神经元, 辅以 B27。板 5 x 104单元格, 每个井到预涂8井室滑动。</li>
	<li>放置在37°c 孵化器2小时的房间幻灯片, 允许细胞附着在底部。然后, 用37摄氏度预热的解冻厘米替换培养基。 注意: 移除培养基并添加收集的 CM 时, 小心不要触及8井室滑动的底部。</li>
	<li>15 h 在初始电镀后, 取出培养基, 用 PBS 清洗细胞一次。然后, 加入200µL 冰冷4% 多聚甲醛溶液的水井, 并孵化细胞与多聚甲醛溶液20分钟在4°c 注: 突起的根背节神经元培养应精确限制为15小时。在这种情况下, 没有可观的突起。</li>
	<li>用冷的 PBS 洗三次, 然后用10% 正常的山羊血清 (anti-Tuj-1) 与0.1% 的海卫 x 30 分钟进行阻断. 孵育固定细胞, 并以 10% (浓度为1µg/毫升) 稀释 4 h 在房间内温度或隔夜在4°c。</li>
	<li>用 PBS 冲洗三次, 然后在室温下用二级抗体 (山羊-抗小鼠) 溶液孵化细胞, 2 小时。然后, 用 PBS 清洗两次, 并使用安装解决方案安装盖玻片 (24 x 50 mm) 的文化幻灯片。</li>
	<li>使用荧光显微镜拍摄图像, 以可视化突起的产物。</li>
</ol>

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作者没有什么可透露的。

这一共同文化体系的产生有几个关键步骤。重要的是要确保小鼠背节神经元和腹腔巨噬细胞的准备新鲜和健康。当所有的 DRGs 的解剖都超过30分钟时, 我们经历了突起的 CM 生长活动。此外, 腹腔巨噬细胞中血液成分的污染也导致了突起在 CM 中的生长活动减少。为了通过 db 阵营诱发强健的神经元巨噬细胞相互作用, 彻底冲洗也将是确保完全清除组织碎片和消除可能残留的细胞毒成分, 如胶原酶或红细胞溶解缓冲物的关键步骤。另一点要重申的是, 应尽可能删除附加到 DRGs 的根。根的残余存根将增加雪旺细胞在背节神经元培养中的污染。雪旺细胞可以产生大量的神经营养因子的文化, 可能混淆的结果。
在这个协议中, 游离巨噬细胞被镀在细胞培养插入物分离的背节神经元镀在底部的井板。我们以前的研究没有显示出显著的差异, 从直接共培养的 CMs (允许两个细胞类型之间的物理接触) 和共培养的突起生长活动的程度, 与两个细胞类型分离单元格区域性插入11。这一结果表明, 神经元和巨噬细胞通过从任何一种细胞类型释放的可溶性分子相互通信, 而不是通过直接的物理接触。结果表明, 神经节神经元分泌的 CCL2, 在这种共培养模型14中, 负责激活巨噬细胞, 使其成为一种支持再生的表型。因此, 我们的共同培养系统将允许澄清更详细的细胞间信号, 调解激活的促再生巨噬细胞。
在本议定书的突起中, 这种培养被精确地限制为15小时。在实验中, 我们研究了几种不同的培养时间, 发现在控制条件 (15 h) 的突起的最小范围内, 高度健壮的突起生长是通过使用 db 营处理的 CM 获得的。小鼠根背节神经元, 如果不预先条件, 不生长任何重要的突起在这个时间框架内。然而, 如果根节神经节神经元的培养时间长于15小时, 它们就会在控制中显示出某种程度的突起, 而不是预先条件, 这将掩盖 db 阵营治疗的 CM 对突起产生的任何影响。在前一项研究中, 使用大鼠背根神经节神经元进行的突起生长试验中也有类似的结果16。
以前的一些研究使用酵母多糖, 酵母细胞壁制备, 激活细胞的支持再生表型 7, 9.然而, 酵母多糖刺激的巨噬细胞不仅释放了支持再生的分子, 而且还发布了细胞毒性因子。用凝胶-过滤色谱法分离酵母多糖治疗巨噬细胞中的蛋白质组分时, 巨噬细胞衍生因子≥ 30 kDa 为细胞毒性, 因子≤ 30 kDa 促进轴突再生 7.此外, 酵母多糖注射液对脊髓可能导致明显死亡的神经元和轴突 9.相比之下, 我们的生成支持再生巨噬细胞的协议显示, 比前一个使用酵母多糖的更具生理学。事实上, 在中心分支受伤后, 对根神经节神经元的 db 注入增强了它们的生长轴突能力, 而没有任何关于细胞损伤的报告17,18。在我们的一系列实验中, 我们从未观察到在应用了 db 治疗后的 CM 后, 在培养过程中, 神经节神经元密度的显著降低。我们推测, 我们的协议允许我们生产完全支持再生的巨噬细胞而不并发神经毒性。因此, 在这里报告的协议和实验模型将有助于识别支持再生巨噬细胞的分子特征, 并通过何种机制来理解促再生型的形成。

在脊髓或脑损伤后, 各种研究试图增强中枢神经轴突再生。炎症反应, 不可避免地伴随着神经系统的损伤, 传统上被认为参与继发病理过程导致有害结果1,2。事实上, 能抑制炎症反应的甲基强的甲泼尼松龙是唯一经批准的治疗急性脊髓损伤的方法3。然而, 最近的研究表明, 有代表性的炎症细胞类型的巨噬细胞可以参与再生或修复受损神经系统的4,5,6。例如, 在晶状体损伤后浸润巨噬细胞产生支持再生分子, 促进视网膜神经节神经元的再生7,8。此外, 移植的背根神经节神经元增加轴突生长的区域, 巨噬细胞由酵母多糖9激活.此外, 病变部位的巨噬细胞可以为受损的周围神经创造一个生长许可的环境, 如10。
我们的研究也提供了强有力的证据, 证明巨噬细胞能促进相邻神经元轴突再生的能力。我们已经表明, 在背根神经节中的巨噬细胞活化是重要的, 以增强再生能力的节神经的感觉神经元后, 预适应周围神经系统损伤11。另一实验室12独立报告了类似的研究。我们还表明, intraganglionic 注射 dibutyryl 循环放大器 (db 阵营), 这是一个众所周知的分子, 以提高轴突再生能力的13, 诱导激活的巨噬细胞。巨噬细胞的失活消除了 db 阵营对突起生长活动的影响。随后的工程鉴定了损伤诱导神经元中 CCL2 的表达, 作为刺激细胞的信号, 可再生表型14,15。
基于上述实验结果, 我们建立了一个体外模型, 类似于在 DRGs 后发生的分子事件, 在预适应损伤模型11,14。在这个模型中, db 阵营被应用于神经元-巨噬细胞共培养诱导细胞间信号, 导致激活的巨噬细胞与支持再生表型。在这里, 我们描述了详细的协议, 我们可以生成巨噬细胞分泌分子因子促进突起的产生 (图 1)。这个实验模型说明了一个概念, 即巨噬细胞可以被刺激或诱导, 以支持轴突再生后的神经系统损伤。我们的模型也将有助于研究细胞间信号的机制, 导致激活的促再生巨噬细胞。

<table><tbody><tr><td>Cell culture insert transparent PET membrane 0.4&amp;mu;m pore size</td><td>Corning,Falcon</td><td>353090</td><td>Transparent PET membrane with 0.4-&amp;mu;m pore size, for 6-well plate</td></tr><tr><td>70-&amp;mu;m nylon cell strainer</td><td>Corning, Falcon</td><td>352350</td><td></td></tr><tr><td>8-well culture slide</td><td>Biocoat</td><td>354632</td><td>with a uniform application of Poly-D-Lysine</td></tr><tr><td>Red blood cell lysis buffer</td><td>Qiagen</td><td>158904</td><td></td></tr><tr><td>Collagenase from &lt;em&gt;Clostridium histolyticum&lt;/em&gt;</td><td>Sigma-Aldrich</td><td>C9407-100MG</td><td></td></tr><tr><td>Neurobasal medium</td><td>Thermo Fisher Scientific, Gibco</td><td>21103-049</td><td>Containing 1% glutamax and 1% penicilin-streptomycin</td></tr><tr><td>B-27 supplement, serum free</td><td>Thermo Fisher Scientific, Gibco</td><td>17504-044</td><td>extracellular solution</td></tr><tr><td>Glutamax</td><td>Thermo Fisher Scientific, Gibco</td><td>35050-061</td><td></td></tr><tr><td>Penicillin-streptomycin</td><td>Thermo Fisher Scientific, Gibco</td><td>15140-122</td><td></td></tr><tr><td>Poly-D-lysine Hydrobromide</td><td>Sigma-Aldrich</td><td>P6407-5MG</td><td></td></tr><tr><td>Laminin</td><td>Thermo Fisher Scientific, Invitrogen</td><td>23017-015</td><td></td></tr><tr><td>Adenosine 3&#039;, 5&#039;-cyclic monophosphate, N&lt;sup&gt;6&lt;/sup&gt;,O2&#039;-dibutyryl-, sodium salt</td><td>Merck Millipore Corporation, Calbiochem</td><td>28745</td><td></td></tr><tr><td>10% Normal Goat Serum</td><td>Thermo Fisher Scientific</td><td>16210072</td><td></td></tr><tr><td>Triton-X-100</td><td>Daejung Chemical and Metal Co</td><td>8566-4405</td><td></td></tr><tr><td>Anti &amp;beta; III tubulin (Tuj-1)</td><td>Promega Corporation</td><td>G7121</td><td>Mouse monoclonal antibody</td></tr><tr><td>Goat anti-Mouse IgG (H+L) secondary antibody</td><td>Thermo Fisher Scientific, Invitrogen</td><td>A11005</td><td></td></tr><tr><td>Hemacytometer</td><td>Marienfeld-Superior</td><td>N/A</td><td></td></tr><tr><td>Cell culture CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Panasonic</td><td>N/A</td><td></td></tr><tr><td>Dissecting stereomicroscope</td><td>Carl Zeiss</td><td>Stemi DV4</td><td></td></tr><tr><td>Twist shaker</td><td>FINEPCR</td><td>Tw3t</td><td></td></tr><tr><td>Tabletop centrifuge</td><td>Sorvall</td><td>N/A</td><td></td></tr><tr><td>Confocal microscope</td><td>Olympus America Inc</td><td>IX71</td><td></td></tr><tr><td>FBS (Fetal Bovine Serum)</td><td>VWR International, Hyclone</td><td>SH30919.03</td><td></td></tr><tr><td>Friedman Pearson Rongeurs</td><td>FST (Fine Science Tools)</td><td>16021-14</td><td>Stainless steel, 14cm, curved, single joint action</td></tr><tr><td>Fine Scissors - Tungsten Carbide &amp;amp; ToughCut</td><td>FST (Fine Science Tools)</td><td>14558-11</td><td>sharp, serrated</td></tr><tr><td>Dumont #7 Forceps</td><td>FST (Fine Science Tools)</td><td>11272-30</td><td>Dumoxel, 0.07 x 0.04mm, curved</td></tr><tr><td>Vannas Spring Scissors</td><td>FST (Fine Science Tools)</td><td>15000-00</td><td>straight, 3mm cutting-edge, sharp</td></tr><tr><td>Qualitron DW-41 Micro-Centrifuge</td><td>Artisan Technology Group</td><td>DW-41</td><td>Input Voltage: 115VAC</td></tr></tbody></table>

neuron-macrophage co-cultures to activate macrophages secreting molecular factors with neurite outgrowth activity

有确凿的证据表明巨噬细胞可以参与损伤神经系统的再生或修复。在这里, 我们描述了一个协议, 其中诱导巨噬细胞产生条件培养基 (CM), 促进突起的生长。成人背根神经节神经元是尖锐的离解和电镀。在神经元稳定附着后, 腹腔巨噬细胞在同一井上被贴上的单元培养物上进行共培养。Dibutyryl 循环放大器 (db 阵营) 适用于24小时的共培养, 之后, 细胞培养插入含有巨噬细胞被移动到另一个井收集 CM 为72小时。CM 从联合文化处理与 db 阵营, 当应用于一个单独成人背根神经节神经元文化, 展示结实突起生长活动。由单一细胞类型组成的单胞型、背根神经节或腹膜巨噬细胞所获得的突起的 CM 没有表现出其生长活性。这表明, 神经元和巨噬细胞之间的相互作用是必不可少的活化巨噬细胞分泌分子因子与突起生长活动成厘米。因此, 我们的共同培养范式也将有助于研究神经元-巨噬细胞相互作用中的细胞间信号, 以刺激巨噬细胞被赋予可再生的表型。

我们描述了一种能产生能分泌突起的分子因子的巨噬细胞的协议。用 db 阵营处理的共培养物获得的巨噬细胞 CM 在应用于单独的背根神经节神经元培养 (图 2A) 时产生了强健的突起。相比之下, 从 PBS 处理的共培养中获得的 CM 并没有在我们的15小时文化持续时间内诱导突起的产生 (图 2B)。当 db 阵营单独对待与巨噬细胞的文化时, 从这种情况得到的 CM 是不有效的支持突起生长 (图 2C)。这表明神经元巨噬细胞相互作用是刺激巨噬细胞被赋予 proregenerative 能力不可或缺的。我们还测试了, 如果 CM 获得从 db 阵营处理的仅神经元的文化, 并没有发现突起的结果 (图 2D)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56920/56920fig1.jpg"> 图 1: 一个示意图, 说明了获取含有突起的条件介质的过程.成人背根背节神经元 (每井 2 x 106细胞) 培养为 4 h。然后, 腹腔巨噬细胞在插入井中培养, 位于神经节神经元培养的井上方。4 h 在电镀以后, PBS 或 dibutyryl 阵营被对待。在24小时的孵化后, 插入井被转移到另一井充满约1ml 介质。然后, 转移井孵化72小时。然后, 收集了孵化条件培养基 (CM)。<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56920/56920fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56920/56920fig2.jpg"> 图 2: 使用条件培养基获得的不同情况下的突起结果.(A、B)成人背根神经节神经元在电镀后完全分离, 培养为15小时2小时, 培养基被条件培养基 (CM) 所取代, 由由 PBS (A) 或 dibutyryl 营 (B) 处理的神经元-巨噬细胞共同培养物所获得。只有用 db 营处理的 CM 表现出强健的突起生长活性。(C、D)从由巨噬细胞 (C) 或神经元 (D) 组成的不同的培养中获得的 CM 不支持突起的生长。刻度条指示200µm.<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56920/56920fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Neuron-Macrophage Co-cultures to Activate Macrophages Secreting Molecular Factors with Neurite Outgrowth Activity at JoVE.com

Neuron-Macrophage Co-cultures to Activate Macrophages Secreting Molecular Factors with Neurite Outgrowth Activity

神经元-巨噬细胞共培养活化巨噬细胞分泌突起活性的分子因子

目前的协议提供了实验程序, 以刺激培养的巨噬细胞, 有能力释放分子因子, 促进突起的生长。对神经元-巨噬细胞共培养的治疗, 诱导巨噬细胞产生具有强突起活性的条件培养基。

There is strong evidence that macrophages can participate in the regeneration or repair of injured nervous system. Here, we describe a protocol in which ...

neuron-macrophage-co-cultures-to-activate-macrophages-secreting

Nanyang Technological University

Research

JoVE Journal

Neuroscience

8.7K 次观看.  Ajou University School of Medicine. 目前的协议提供了实验程序, 以刺激培养的巨噬细胞, 有能力释放分子因子, 促进突起的生长。对神经元-巨噬细胞共培养的治疗, 诱导巨噬细胞产生具有强突起活性的条件培养基。

视频: Neuron-Macrophage Co-cultures to Activate Macrophages Secreting Molecular Factors with Neurite Outgrowth Activity

Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased understanding of the molecular events that give rise to glioblastomas, this cancer still remains highly refractory to conventional treatment. Surgical resection of high grade brain tumors is rarely complete due to the highly infiltrative nature of glioblastoma cells. Therapeutic approaches which attenuate glioblastoma cell invasion therefore is an attractive option. Our laboratory and others have shown that tumor associated macrophages and microglia (resident brain macrophages) strongly stimulate glioblastoma invasion. The protocol described in this paper is used to model glioblastoma-macrophage/microglia interaction using in vitro culture assays. This approach can greatly facilitate the development and/or discovery of drugs that disrupt the communication with the macrophages that enables this malignant behavior. We have established two robust coculture invasion assays where microglia/macrophages stimulate glioma cell invasion by 5 - 10 fold. Glioblastoma cells labelled with a fluorescent marker or constitutively expressing a fluorescent protein are plated without and with macrophages/microglia on matrix-coated polycarbonate chamber inserts or embedded in a three dimensional matrix. Cell invasion is assessed by using fluorescent microscopy to image and count only invasive cells on the underside of the filter. Using these assays, several pharmacological inhibitors (JNJ-28312141, PLX3397, Gefitinib, and Semapimod), have been identified which block macrophage/microglia stimulated glioblastoma invasion.

Understanding the malignant behavior of cancer requires creating accurate models of how tumor cells interact with components of the tumor microenvironment, such as macrophages. Here we describe two methods to study glioblastoma cell interaction with tumor associated macrophages and microglia where the effect on glioblastoma invasion is assessed.

共培养化验，以研究胶质母细胞瘤入侵的巨噬细胞和小胶质细胞刺激

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased ...

科研

癌症研究

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their antigen presentation and T cell activation capacities. Since most vaccinations of experimental tumor models require millions of DC, they are widely isolated from the bone marrow or spleen. However, these DC significantly differ from blood and tumor DC in their responses to immune complexes (IC), and presumably to other Syk-coupled lectin receptors. Importantly, given the sensitivity of DC to danger-associated molecules, the presence of endotoxins or antibodies that crosslink activation receptors in one of the isolating steps could result in the priming of DC and thus affect the parameters, or at least the dosage, required to activate them. Therefore, here we describe a detailed protocol for isolating MoDC from blood and tumors while avoiding their premature activation. In addition, a protocol is provided for MoDC activation with tumor IC, and their subsequent analyses.

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Monocyte-derived DC (MoDC) can sense minor amounts of danger-associated molecules and are therefore easily primed. We provide a detailed protocol for the isolation of MoDC from blood and tumors and their activation with immune complexes while highlighting key precautions that should be considered in order to avoid their premature activation.

小鼠单核细胞衍生树突状细胞的分离及与肿瘤免疫配合物的后体外活化

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their ...

Studying the Effects of Tumor-Secreted Paracrine Ligands on Macrophage Activation using Co-Culture with Permeable Membrane Supports

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer growth and metastasis. Paracrine signals can involve cell-cell contact between different cell types, such as PD-L1 expressed on the surface of tumors interacting directly with PD-1 on the surface of T cells, or the secretion of ligands by a tumor cell to affect an immune cell. Here we describe a co-culture method to interrogate the effects of tumor-secreted ligands on immune cell (macrophage) activation. This straightforward procedure utilizes commercially available 0.4 &#181;m polycarbonate membrane permeable supports and standard tissue culture plates. In the process described, macrophages are cultured in the upper chamber and tumor cells in the lower chamber. The presence of the 0.4 &#181;m barrier allows for the study of intercellular signaling without the confounding variable of physical contact, because the two cell types share the same medium and exposure to paracrine ligands. This approach can be combined with others, such as genetic alterations of the macrophage (e.g., isolation from genetic knock-out mice) or tumor (e.g., CRISPR-mediated alterations) to study the role of specific secreted factors and receptors. The approach also lends itself to standard molecular biological analyses such as quantitative reverse transcription polymerase chain reaction (qRT-PCR) or Western blot analysis, without the need for flow sorting to separate the two cell populations. Enzyme-linked immunosorbent assays (ELISAs) can similarly be utilized to measure secreted ligands to better understand the dynamic interaction of cell signaling in the multiple cell type context. Duration of co-culture can also be varied for the study of temporally regulated events. This co-culture method is a robust tool that facilitates the study of tumor-secreted signals in the immune context.

Here, we present a method using permeable membrane supports to facilitate the study of non-contact paracrine signaling used by tumor cells to suppress the immune response. The system is amenable to studying the role of tumor-secreted factors in dampening macrophage activation.

研究肿瘤分泌性副体结扎对宏噬细胞活化的影响，使用可渗透膜支架共培养

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer ...

An Engulfment Assay: A Protocol to Assess Interactions Between CNS Phagocytes and Neurons

Phagocytosis is a process in which a cell engulfs material (entire cell, parts of a cell, debris, etc.) in its surrounding extracellular environment and subsequently digests this material, commonly through lysosomal degradation. Microglia are the resident immune cells of the central nervous system (CNS) whose phagocytic function has been described in a broad range of conditions from neurodegenerative disease (e.g., beta-amyloid clearance in Alzheimer&#8217;s disease) to development of the healthy brain (e.g., synaptic pruning)1-6. The following protocol is an engulfment assay developed to visualize and quantify microglia-mediated engulfment of presynaptic inputs in the developing mouse retinogeniculate system7. While this assay was used to assess microglia function in this particular context, a similar approach may be used to assess other phagocytes throughout the brain (e.g., astrocytes) and the rest of the body (e.g., peripheral macrophages) as well as other contexts in which synaptic remodeling occurs (e.g. ,brain injury/disease).

Microglia are the resident immune cells of the central nervous system (CNS) with a high capacity to phagocytose or engulf material in their extracellular environment. Here, a broadly applicable, reliable, and highly quantitative assay for visualizing and measuring microglia-mediated engulfment of synaptic components is described.

一个吞噬含量：一个协议，以评估之间的中枢神经系统吞噬细胞和神经元的相互作用

Phagocytosis is a process in which a cell engulfs material (entire cell, parts of a cell, debris, etc.) in its surrounding extracellular environment and ...

神经科学

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. ...

A Macrophage Reporter Cell Assay to Examine Toll-Like Receptor-Mediated NF-kB/AP-1 Signaling on Adsorbed Protein Layers on Polymeric Surfaces

The persistent inflammatory host response to an implanted biomaterial, known as the foreign body reaction, is a significant challenge in the development and implementation of biomedical devices and tissue engineering constructs. Macrophages, an innate immune cell, are key players in the foreign body reaction because they remain at the implant site for the lifetime of the device, and are commonly studied to gain an understanding of this detrimental host response. Many biomaterials researchers have shown that adsorbed protein layers on implanted materials influence macrophage behavior, and subsequently impact the host response. The methods in this paper describe an in vitro model using adsorbed protein layers containing cellular damage molecules on polymer biomaterial surfaces to assess macrophage responses. An NF-&#1082;B/AP-1 reporter macrophage cell line and the associated colorimetric alkaline phosphatase assay were used as a rapid method to indirectly examine NF-&#1082;B/AP-1 transcription factor activity in response to complex adsorbed protein layers containing blood proteins and damage-associated molecular patterns, as a model of the complex adsorbed protein layers formed on biomaterial surfaces in vivo.

This protocol provides researchers with a rapid, indirect method of measuring TLR-dependent NF-&#1082;B/AP-1 transcription factor activity in a murine macrophage cell line in response to a variety of polymeric surfaces and adsorbed protein layers that model the biomaterial implant microenvironment.

巨噬细胞报告器细胞分析，以检查聚合物表面的吸附蛋白层上的收费类似受体介导NF-kB/AP-1信号

The persistent inflammatory host response to an implanted biomaterial, known as the foreign body reaction, is a significant challenge in the development ...

生物工程

In vitro Quantitative Imaging Assay for Phagocytosis of Dead Neuroblastoma Cells by iPSC-Macrophages

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson&#39;s disease and Alzheimer&#39;s disease. Microglia clear up dead and dying neurons through the process of efferocytosis, a specialized form of phagocytosis. The phagocytosis function can be disrupted by environmental or genetic risk factors that affect microglia. This paper presents a rapid and simple in vitro&#160;microscopy protocol for studying microglial efferocytosis in an induced pluripotent stem cell (iPSC) model of microglia, using a human neuroblastoma cell line (SH-SY5Y) labeled with a pH-sensitive dye for the phagocytic cargo. The procedure results in a high yield of dead neuroblastoma cells, which display surface phosphatidylserine, recognized as an &#34;eat-me&#34; signal by phagocytes. The 96-well plate assay is suitable for live-cell time-lapse imaging, or the plate can be successfully fixed prior to further processing and quantified by high-content microscopy. Fixed-cell high-content microscopy enables the assay to be scaled up for screening of small molecule inhibitors or assessing the phagocytic function of genetic variant iPSC lines. While this assay was developed to study phagocytosis of whole dead neuroblastoma cells by iPSC-macrophages, the assay can be easily adapted for other cargoes relevant to neurodegenerative diseases, such as synaptosomes and myelin, and other phagocytic cell types.

Neurodegenerative diseases are associated with dysregulated microglia functions. This article outlines an in vitro assay of phagocytosis of neuroblastoma cells by iPSC-macrophages. Quantitative microscopy readouts are described for both live-cell time-lapse imaging and fixed-cell high-content imaging.

iPSC-巨噬细胞死亡神经母细胞的体外定量成像检测

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson&#39;s disease and Alzheimer&#39;s disease. ...

Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt to make straightforward the very complex relationships between the several cellular subtypes that compose multicellular organisms, in vitro techniques have been developed to help researchers acquire a detailed understanding of single cell populations. One of these techniques uses inserts with a permeable membrane allowing secreted soluble factors to diffuse. Thus, a population of cells grown in inserts can be co-cultured in a well or dish containing a different cell type for evaluating cellular changes following paracrine signaling in the absence of cell-cell contact. Such insert co-culture systems offer various advantages over other co-culture techniques, namely bidirectional signaling, conserved cell polarity and population-specific detection of cellular changes. In addition to being utilized in the field of inflammation, cancer, angiogenesis and differentiation, these co-culture systems are of prime importance in the study of the intricate relationships that exist between the different cellular subtypes present in the central nervous system, particularly in the context of neuroinflammation. This article offers general methodological guidelines in order to set up an experiment in order to evaluating cellular changes mediated by secreted soluble factors using an insert co-culture system. Moreover, a specific protocol to measure the neuroinflammatory effects of cytokines secreted by lipopolysaccharide-activated N9 microglia on neuronal PC12 cells will be detailed, offering a concrete understanding of insert co-culture methodology.

In multicellular organisms, secreted soluble factors elicit responses from different cell types as a result of paracrine signaling. Insert co-culture systems offer a simple way to assess the changes mediated by secreted soluble factors in the absence of cell-cell contact.

在细胞间接触缺失的两个蜂窝型的插入共培养系统的开发

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt ...

生物学

 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

Generating and Co-culturing Murine Primary Microglia and Cortical Neurons

Microglia are tissue-resident macrophages of the central nervous system (CNS), performing numerous functions that support neuronal health and CNS homeostasis. They are a major population of immune cells associated with CNS disease activity, adopting&#160;reactive phenotypes that potentially contribute to neuronal injury during chronic neurodegenerative diseases such as multiple sclerosis (MS). The distinct mechanisms by which microglia regulate neuronal function and survival during health and disease remain limited due to challenges in resolving the complex in vivo interactions between microglia, neurons, and other CNS environmental factors. Thus, the in vitro approach of co-culturing microglia and neurons remains a valuable tool for studying microglia-neuronal interactions. Here, we present a protocol to generate and co-culture primary microglia and neurons from mice. Specifically, microglia were isolated after 9-10 days in vitro from a mixed glia culture established from brain homogenates derived from neonatal mice between post-natal days 0-2. Neuronal cells were isolated from brain cortices of mouse embryos between embryonic days 16-18. After 4-5 days in vitro, neuronal cells were seeded in 96-well plates, followed by the addition of microglia to form the co-culture. Careful timing is critical for this protocol as both cell types need to reach experimental maturity to establish the co-culture. Overall, this co-culture can be useful for studying microglia-neuron interactions and can provide multiple readouts, including immunofluorescence microscopy, live imaging, as well as RNA and protein assays.

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

This protocol describes a microglia-neuronal co-culture established from primary neuronal cells isolated from mouse embryos at embryonic days 15-16 and primary microglia generated from the brains of neonatal mice at post-natal days 1-2.