作者想感谢苏联医学院的媒体专家, 他在视频制作、编辑和配音方面的工作。
这项工作得到了国家卫生研究院 (R01GM100474 和 R01GM072611) 的支持。

在这里和2节中描述的方法已得到佛罗里达州立大学动物保育和使用委员会 (IACUC) 的批准, 并遵循《实验室动物护理和使用指南》中规定的指南, 8th版.在这个协议中使用的所有动物都是在一个专门的实验室动物设施的房子, 直到使用。没有在体内试验是在牺牲之前进行的。动物数量是根据实验需要使用平均细胞和髓鞘收集作为指导, 以尽量减少使用。
注意:该协议描述了生成骨髓巨噬细胞 (BMDMs) (1 节), 荧光标记脑源性髓鞘碎片的制备 (2 节), 分析髓鞘碎片吞噬的一般程序 (第3节) 和髓鞘堆积物脂质积累分析的一般程序 (4 节)。在层流生物安全柜中, 应完成试剂制备、细胞采集和操作、髓鞘的收集和标记、化验性能的测定。
1. 原发骨髓巨噬细胞的生成
<ol>
	<li>全巨噬细胞培养基的制备 (CMCM) 注:骨髓巨噬细胞的生成需要使用巨噬细胞集落刺激因子 (M-CSF)。本制剂利用 L929 小鼠成纤维细胞条件培养基作为 M 型脑脊液的来源。<ol>
		<li>用50毫升高葡萄糖 (4500 毫克/l-葡萄糖) 培养细胞在145毫米菜肴中生成 L929 小鼠成纤维细胞条件培养基 (DMEM) Dulbecco 的改良鹰培养基补充5% 新出生的小牛血清和青霉素/链霉素7天。</li>
		<li>收集培养基从文化到50毫升圆锥离心管和离心机30分钟在 3500 x g, 4 ° c。</li>
		<li>过滤器清通过0.2 µm 注射器过滤器进入一个新的50毫升圆锥离心管。被适应的媒介可以被存放在-80 ° c 为6月。</li>
		<li>对高糖 DMEM, 增加5% 卷/卷的非华语, 15% 卷/卷 L929 小鼠成纤维细胞条件 DMEM, 1% 青霉素/链霉素。介质可储存在4° c 2-3 月。使用前要预热到37° c。</li>
	</ol>
	</li>
	<li>原发性骨髓细胞的采集与巨噬细胞诱导 
	注意:使用这个协议一只老鼠将产生大约2000万骨髓来源的巨噬细胞 (BMDMs)。<ol>
		<li>安乐使用标准 CO2窒息指南的8-10 周老鼠的期望数量, 其次是颈椎脱位。</li>
		<li>使用 70% (卷/卷) 乙醇对动物进行消毒: H2O, 使毛皮饱和。</li>
		<li>在腹部切开一个小开口, 并小心地拉回皮肤, 露出底层的组织。注意不要穿刺腹膜腔。</li>
		<li>从臀部开始, 在脚踝处切除两条后腿。将所收集的组织放入100毫米的培养皿中, 其中含有5-10 毫升无菌磷酸缓冲盐水 (PBS), 辅以1% 青霉素/链霉素。 
		注意:当处理几个动物时, 一定要保持在冰上的菜肴, 以限制组织退化。</li>
		<li>用手术刀从骨头上取出肌肉和结缔组织。只有股骨和胫骨用于细胞收集, 腓骨可以被丢弃。</li>
		<li>通过用手术刀将每个末端的小部分修剪, 使收集到的骨骼露出骨髓腔。</li>
		<li>使用25g 针, 冲洗骨髓腔与 CMCM 成一个50毫升圆锥离心管。一旦足够冲洗, 骨骼就会呈白色。</li>
		<li>一旦收集完成, 煽动物与18g 针30-90 秒, 产生一个单一的细胞悬浮。 
		注意:可选红细胞 (红细胞) 裂解步骤可在这一点上进行。最近有人建议, 细胞内铁含量可能影响髓鞘碎片对巨噬细胞极化的影响16。有关包含红细胞裂解步骤的信息, 请参阅 Trouplin et al。17。</li>
		<li>将悬浮液通过70µm 无菌细胞过滤器滤入新的50毫升锥形离心管中。</li>
		<li>种子收集的细胞均匀地成145毫米细胞培养皿包含15-20 毫升 CMCM。每只老鼠使用大约三培养皿。孵育文化在37° c, 5% CO2。</li>
		<li>经过72小时洗板后用无菌 PBS 去除换药细胞, 然后添加15-20mL 新鲜 CMCM。孵育文化为另外4天在37° c, 5% CO2。经过7天的总培养, 最初孤立的造血骨髓细胞将是成熟的 BMDMs (图 1)。</li>
	</ol>
	</li>
</ol>
2. 荧光标记脑源性髓鞘碎片的生成
注意:所有试剂可贮存在4° c, 可达1月。
<ol>
	<li>髓鞘碎片收集试剂的制备
	<ol>
		<li>准备 Tris·Cl 缓冲液。<ol>
			<li>到800mL 蒸馏去离子 H2o (ddiH2o) 添加 20 mL 1 米 Tris·Cl, pH 值 7.45 (20 mm 最终浓度) 和20毫升100毫米 Na2EDTA (2 毫米最终浓度)。</li>
			<li>调整 pH 值至7.45。通过0.2 µm 过滤单元, 通过 ddiH2o. 过滤器解决方案将音量调整为1000毫升。</li>
		</ol>
		</li>
		<li>准备1米蔗糖溶液。<ol>
			<li>到100毫升的 Tris·Cl 缓冲液, 加68.46 克蔗糖。用 Tris·调节音量至200毫升Cl 缓冲液。过滤器解决方案通过0.2 µm 过滤装置。</li>
		</ol>
		</li>
		<li>用 Tris·稀释 1 m 溶液, 制备200毫升的0.32 米蔗糖溶液Cl 缓冲器解决方案 0.83:0. 16 (卷/卷)。</li>
		<li>用 Tris·稀释 1 m 溶液, 制备150毫升的0.83 米蔗糖溶液Cl 缓冲器解决方案 0.83:0. 16 (卷/卷)。</li>
	</ol>
	</li>
	<li>收集脑衍生的粗髓鞘碎片 
	注意:这里描述的收集方法是为了隔离粗髓鞘碎片。<ol>
		<li>安乐10-12 只小鼠8-10 周龄使用标准 CO2窒息指南后, 颈椎脱位。</li>
		<li>解剖的大脑和放置在一个100毫米的菜, 其中含有10毫升的0.32 米蔗糖溶液。</li></ol></li></ol>把盘子放在冰上。
		<li>使用无菌手术剪刀将大脑切成大约 5 mm3大小的碎片。</li>
		<li>将组织转移到50毫升圆锥离心管上, 加入约30毫升的0.32 米蔗糖溶液。</li>
		<li>质与无菌 hand-held 旋转均质机, 直到平滑的解决方案实现。</li>
		<li>用0.32 米蔗糖溶液将匀浆的大脑稀释到90毫升的最终体积。</li>
		<li>对六38.5 毫升的薄壁聚丙烯 ultracentrifuge 管, 加入20毫升的0.83 米蔗糖溶液。</li>
		<li>轻轻地在0.83 米蔗糖溶液的顶端加入均匀化的大脑溶液, 注意不要混合两层。</li>
		<li>平衡每管与0.32 米蔗糖溶液。</li>
		<li>离心机在 10万 x g 为45分钟, 在4° c 使用适当的预 ultracentrifuge 转子。将转子的加速度和减速设置为最小值, 以减少髓鞘碎片的损失。</li>
		<li>从两个蔗糖密度的界面收集髓磷脂碎片。 
		注意: 碎片应显示为一个白色的带向中心的管。</li>
		<li>将粗髓鞘碎片组合成50毫升锥形离心管, 并使用 Tris·将体积调整到大约35毫升。Cl 缓冲液。</li>
		<li>质的粗髓鞘碎片与无菌 hand-held 旋转均质机 30-60 s。</li>
		<li>均匀地划分6干净的 ultracentrifuge 管之间的悬浮和平衡与适当的容量 Tris·Cl 缓冲液。</li>
		<li>离心机在 10万 x g 为45分钟, 在4° c 使用适当的预 ultracentrifuge 转子。将转子加速和减速设置为其最大值。</li>
		<li>由于固体白色小球现在将是可见的, 丢弃上清和 re-suspend 10-15 毫升的 Tris·颗粒Cl 缓冲液。</li>
		<li>均匀地划分2干净的 ultracentrifuge 管之间的悬浮和平衡与适当的容量 Tris·Cl 缓冲液。</li>
		<li>再次离心在 10万 x g 为45分钟在4° c 使用适当的预 ultracentrifuge 转子。将转子加速和减速设置为其最大值。</li>
		<li>丢弃上清和重5-6 毫升无菌 PBS 中的颗粒, 并在适当数量的称量1.5 毫升微离心管之间划分悬浮。</li>
		<li>离心机在 2.2万 x g 为10分钟在4° c。</li>
		<li>丢弃上清液, 确定髓鞘碎片颗粒的重量。</li>
		<li>将 PBS 中的小球再悬浮到100毫克/毫升的最后浓度。 
		注意: 十到十二的大脑足以产生10-15 毫升的100毫克/毫升髓鞘碎片。髓鞘碎片可贮存在-80 ° c 6 月。</li>
	
	
	<li>髓鞘碎片荧光标记
	<ol>
		<li>在使用前立即准备50µM 羧基琥珀酰亚胺酯 (CFSE) 溶液。<ol>
			<li>使用无菌 PBS, 稀释5毫米的 CFSE, 用100% 二甲基亚砜 (甲基亚砜) 制备的溶液, 最终工作浓度为50µM。</li>
			<li>过滤器解决方案通过0.2 µm 注射器过滤器。</li>
		</ol>
		</li>
		<li>解冻所需的100毫克/毫升髓鞘碎片和 re-suspend 与无菌29g 针。 
		注意: 在使用前, 冻结髓鞘碎片会导致它从需要悬浮的溶液中掉出来。</li>
		<li>将髓鞘碎片转移到称量1.5 毫升微离心管。</li>
		<li>离心机在 14800 x g 为10分钟在4° c。丢弃上清液。</li>
		<li>重200µL CFSE 溶液中的髓鞘碎片, 每100µL 髓鞘碎片颗粒。</li>
		<li>在室温下孵育30分钟 (RT), 免受光线的保护。</li>
		<li>离心机在 14800 x g 为10分钟在4° c。丢弃上清液。</li>
		<li>重在600-800 µL 的洗涤缓冲器 (0.2 µm 过滤器灭菌100毫米甘氨酸在 PBS) 的颗粒。</li>
		<li>离心机在 14800 x g 为10分钟在4° c。丢弃上清液。</li>
		<li>重复步骤 2.3. 8-2. 3.9 两次。</li>
		<li>最后洗涤后, 确定髓鞘碎片颗粒和 re-suspend 的重量100毫克/毫升无菌 PBS。 注: 带标记的髓鞘碎片可贮存在-80 ° c, 可长达6月。</li>
	</ol>
	</li>

3. 髓鞘碎片吞噬试验
注意:下面是观察荧光标记髓鞘碎片吞噬功能的基本方法。其他的治疗和实验条件的增加将需要由调查员优化。
<ol>
	<li>处理板的制备
	<ol>
		<li>对于每145毫米板, 收集成熟的 BMDMs 成一个15毫升圆锥离心管使用5-6 毫升的10毫米乙酸酸 (EDTA) 在 Dulbecco 的磷酸盐缓冲盐水 (dPBS) (pH 7.4)。 
		注意:不要使用胰蛋白酶, 因为它可能会改变细胞的活化状态18。</li>
		<li>在收集的细胞中加入等量的 5ml CMCM。</li>
		<li>离心机在 180 x g 为8分钟在20° c。丢弃上清液。</li>
		<li>重新挂起 CMCM 中的单元格并进行计数。</li>
		<li>对24井细胞培养板的每一个井, 在1毫升的 CMCM 中添加 1x105细胞。</li>
		<li>孵育在37° c, 5% CO2为 24 h。</li>
	</ol>
	</li>
	<li>髓鞘碎片的处理与分析
	<ol>
		<li>添加10µL 100 毫克/毫升 CFSE 标记髓鞘碎片每井 (1 毫克/毫升最终浓度)。</li>
		<li>孵育1-3 小时, 在37° c, 5% CO2。</li>
		<li>洗盘子3次, 用无菌 PBS 去除 non-engulfed 髓鞘碎片。</li>
		<li>每井加400µL 4% 甲醛。在 RT 处孵育30分钟。</li>
		<li>洗涤板2次, 无菌 PBS。</li>
		<li>每井加400µL 赫斯特33258溶液。孵育5分钟在 RT 保护免受光。</li>
		<li>用无菌 PBS 冲洗盘子2次。</li>
		<li>添加200µL 的氟凝胶与三缓冲器, 以减少漂白。</li>
		<li>使用倒置的 epi 荧光显微镜的图像细胞。 
		注意:CFSE 的激发和发射波长分别为 494 nm 和 521 nm。</li>
		<li>将 CFSE 阳性细胞数除以细胞核数, 以确定相对髓鞘碎片的摄取量。</li>
		<li>在成像之前, 保持板长达7天, 在4° c 保护免受光。</li>
	</ol>
	</li>
</ol>
4. 通过油红 O 染色定量测定胞内脂质
注意:下面是观察细胞内髓鞘-碎片衍生脂质的基本方法。由于光谱重叠, 不推荐使用 CFSE 标记的髓鞘碎片进行荧光定量。其他的治疗和实验条件的增加将需要由调查员优化。
<ol>
	<li>染色液的制备
	<ol>
		<li>准备0.5% 油红 O (破产) 染色液。<ol>
			<li>在搅拌时, 缓慢地将100毫升100% 丙二醇 (12-丙醇) 添加到0.5 克 (1-([4-Xylylazo) 二甲苯]-2-萘酚)。</li>
			<li>热溶液在95° c 为 15 min 或直到所有大微粒被溶化。 
			注意: 不要加热超过100° c。</li>
			<li>过滤器解决方案通过一块滤纸, 而仍然温暖到一个新的容器。</li>
			<li>允许解决方案在 rt 中隔夜冷却. 解决方案可在 rt 中存储长达1年。</li>
		</ol>
		</li>
		<li>准备85% 丙二醇溶液。<ol>
			<li>加入85毫升的100% 丙二醇到15毫升的 ddiH2o 搅拌至混合。解决方案可在 RT 中存储多达1年。</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>处理板的制备
	<ol>
		<li>按照3节中概述的方法准备一个24井板。</li>
		<li>孵育在37° c, 5% CO2为 24 h。</li>
	</ol>
	</li>
	<li>髓鞘碎片治疗
	<ol>
		<li>每井添加10µL 100 毫克/毫升髓鞘碎片 (1 毫克/毫升最终浓度)。</li>
		<li>孵育1-3 小时, 在37° c, 5% CO2。</li>
		<li>洗盘子3次, 用无菌 PBS 去除 non-digested 髓鞘碎片。 注意: 在这一点上, 盘子可以立即进行固定或新鲜的 CMCM 可以添加, 细胞返回到孵化额外的时间点。</li>
		<li>每井加400µL 4% 甲醛。在 RT 处孵育30分钟。</li>
		<li>洗涤板2次, 无菌 PBS 然后增益染色。</li>
	</ol>
	</li>
	<li>破产法染色及分析
	<ol>
		<li>用 ddiH2O 清洗固定板3次。</li>
		<li>每井加400µL 100% 丙二醇, 孵育5分钟。 
		注意: 这将减少 ddiH2O 的结转。</li>
		<li>吸入丙二醇, 并在每口井中加入400µL 的破产解决方案。</li>
		<li>孵育8分钟, 在60° c。</li>
		<li>吸入破产解决方案。然后, 每井加400µL 85% 丙二醇, 在 RT 处孵育 5 minumintes。</li>
		<li>洗涤板3次 ddiH2O。</li>
		<li>每井加400µL 赫斯特33258溶液。在 RT 中孵育5分钟, 免受光照。</li>
		<li>用无菌 PBS 冲洗盘子2次。</li>
		<li>添加200µL 的氟凝胶与三缓冲每井。</li>
		<li>使用倒置的 epi 荧光显微镜的图像细胞。破产人可以使用标准的德州红或 DsRed 过滤器集成像。</li>
		<li>通过确定每个图像场中的破产人阳性面积并将其除以细胞核数来量化相对脂质潴留。</li>
		<li>在4° c 保护免受光照的7天内保持板材。</li>
	</ol>
	</li>
</ol>

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作者没有披露。

这里描述的程序利用了新分离的原始的中枢神经系统髓鞘碎片和原发骨髓巨噬细胞。为了减少动物的开支, 我们建议在牺牲时从每只老鼠身上采集大脑和骨髓细胞。两位研究人员一起工作可以同时准备两种材料。另外, 在-80 ° c 的 PBS 中, 大脑可以在髓鞘碎片隔离之前用抗生素补充。我们的经验是, 大脑可以在这些条件下维持1月, 而不会有明显的髓鞘碎片生成潜能的损失。
CFSE 被用来标记髓鞘碎片, 因为它的易用性和荧光灯强度。染料保持淬火, 直到细胞酯在分子14上切割醋酸基团。被释放的琥珀酰亚胺酯基团可以与初级酰胺反应, 从而产生共价键附着到细胞蛋白14。虽然 CFSE 是易激动的使用标准的绿色荧光通道, 有几种衍生物商业可利用不同的频谱属性。因此, 有可能设计一个类似的吞噬试验, 使用多种荧光染料。还应该指出的是, 由于染料很容易穿过细胞膜, 它可以用类似的方式将凋亡细胞标记为髓鞘碎片19。此外, 虽然我们的方法依赖于标准的图像分析, 与用户的优化, 量化可以使用流式细胞仪或高通量板成像系统进行。
油红色 O (也称为溶剂红 27, 或苏丹红 5B) 是一种 fat-soluble 染料, 已广泛用于染色中性脂质的组织学标本。其深红色的色素沉着, 通过明亮的领域和荧光显微镜5,20, 使细胞内脂质可视化。使用荧光显微镜可以进行单通道成像和可靠的量化的破产人染色脂滴。染色方法简单, 重现性好, 在样品制备过程中必须注意。重要的是, 酒精或酸固定不使用, 因为他们可以溶解细胞内脂质, 导致显着减少染色强度。此外, 与任何定量图像分析一样, 标准化的图像捕获设置和足够的无偏置采样的重要性是不能低估的。建议使用染色控制来解释任何样品荧光。这种染色控制应一视同仁, 但不与破产管理署, 以衡量背景荧光的水平。即便如此, 我们的图像分析方法需要比其他的脂质量化技术更少的优化, 如破产法的提取方法21, 这需要标准曲线准备, 更容易实验性的变化, 并要求样品的破坏。基于图像的分析方法在传统的破产处理中具有一些优点, 因为它不具有破坏性, 并且与免疫2122兼容。
最后, 虽然所描述的程序是相对直接的, 这是至关重要的是, 适当的无菌技术使用, 因为巨噬细胞表达的多个受体, 承认病原体相关的分子模式 (PAMPS), 如内毒素脂多糖 (LPS)23。与细菌污染的相互作用会导致巨噬细胞的活化, 并能戏剧性地改变它们的功能24,25。关于收集到的髓鞘碎片, 建议进行污染测试。内毒素检测方法, 如鲎试剂 (拉尔) 测定法具有较高的灵敏度, 并已用于此应用程序的5,19。
巨噬细胞在健康和疾病中起着重要作用, 但 BMDMs 在中枢神经系统损伤中的作用目前是一个重要的研究课题。使用这里描述的方法, 研究人员可以开始更好地了解髓鞘碎片吞噬的机制 BMDMs。

骨髓源性巨噬细胞 (BMDMs) 是先天和适应性免疫系统之间的重要联系。作为抗原呈递细胞 (apc), 他们可以通过抗原呈递和细胞因子释放1,2,3与淋巴细胞进行通信。然而, 作为专业吞噬, 他们的主要功能是清除病原体, 衰老细胞和细胞碎片1,4。继脊髓损伤 (SCI), 大量髓鞘碎片是由死亡的突, 细胞类型负责中枢神经系统轴突鞘5。我们和其他人已经表明, 清除髓鞘碎片主要是渗透 BMDMs 的责任5,6,7。然而, 在脊髓损伤部位吞没髓鞘碎片被建议将这些正常的抗炎细胞转变为炎状态5,8,9。BMDMs 是脊髓损伤中神经炎症的重要介质, 是临床的主要靶点。
为了帮助研究 BMDMs 在损伤脊髓中的影响, 我们开发了一个体外模型, 直接探讨 BMDMs 对髓鞘碎片的反应。为了提高生物学相关性, 在这些调查中使用了小鼠 BMDMs 和新近分离的髓鞘碎片。因此, 这里介绍的方法也详述了原小鼠 BMDMs 的分离和培养, 以及用于分离鼠中枢神经系统衍生髓鞘碎片的改良蔗糖梯度技术10,11,12。髓鞘碎片可以很容易地标记为荧光染料, 羧基琥珀酰亚胺酯 (CFSE), 以跟踪其内部化的 BMDMs. CFSE 是非常适合这个应用, 因为它是 non-cytotoxic, 其狭窄的荧光光谱允许多路传输与其他荧光探针13,14。继吞噬后, 髓鞘碎片脂通过溶酶体转运, 并以中性脂质包装成细胞内脂滴5。为了量化这种细胞内脂质的积累, 我们提出了一个油红 O (破产) 染色方法优化定量图像分析。这个简单的染色方法产生可靠的重现性结果和量化的15。这些方法有助于研究的髓鞘碎片吞噬和脂质保留与有限的专用设备。

<table><tbody><tr><td>DMEM</td><td>GE Healthcare Life Sciences</td><td>SH30243.01</td><td>High glucose with L-glutamine, sodium pyruvate</td></tr><tr><td>Penicillin-Streptomycin Solution</td><td>Corning</td><td>30-002-CI</td><td>100X Solution</td></tr><tr><td>New Born Calf Serum (NCS)</td><td>Rocky Mountain Biologics</td><td>NBS-BBT-5XM</td><td>United States Origin</td></tr><tr><td>NCTC clone 929 [L cell, L-929, derivative of Strain L]&amp;nbsp;</td><td>ATCC</td><td>CCL-1</td><td>L929 Cell Line of Conditioned Media Preparation</td></tr><tr><td>24-well Cell Culture Plates</td><td>VWR</td><td>10062-896&amp;nbsp;</td><td></td></tr><tr><td>Cell Culture Dish</td><td>Greiner Bio-One</td><td>639960</td><td>Polystyrine, 145/20mm</td></tr><tr><td>CFSE Cell Proliferation Kit</td><td>Thermo Fisher</td><td>C34570</td><td>DMSO for Reconsitution Provided</td></tr><tr><td>Fluoro-gel with Tris Buffer&amp;nbsp;</td><td>Electron Microscopy Sciences</td><td>17985-11</td><td></td></tr><tr><td>Oil Red O</td><td>Sigma Aldrich</td><td>O0625</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Materials&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>Ultracentrifuge Tubes</td><td>Beckman Coulter</td><td>326823</td><td>Thinwall, Polypropylene, 38.5 mL, 25 x 89 mm</td></tr><tr><td>SW 32 Ti Ultracentrifuge Rotor</td><td>Beckman Coulter</td><td>369650</td><td>SW 32 Ti Rotor, Swinging Bucket, Titanium</td></tr><tr><td>Hand Held Rotary Homogenizer</td><td>Fisher Science</td><td>08-451-71</td><td></td></tr></tbody></table>

in vitro phagocytosis of myelin debris by bone marrow-derived macrophages

骨髓源性巨噬细胞 (BMDMs) 是成熟的白细胞, 具有重要的生理作用, 作为专业吞噬能够清除各种微粒。正常情况下, BMDMs 受中枢神经系统 (CNS) 的限制, 但在受伤后, 他们可以很容易地渗入。一旦在受损的中枢神经组织内, BMDMs 是主要的细胞类型, 负责清除损伤衍生的细胞碎片, 包括大量的富含脂质的髓鞘碎片。中枢神经系统内 BMDM 浸润和髓鞘碎片吞噬的病理影响是复杂的, 不太清楚。这里描述的协议, 允许直接的体外研究 BMDMs 在中枢神经系统损伤的情况下。我们覆盖小鼠 BMDM 分离和培养, 髓鞘碎片制备, 并测定 BMDM 髓鞘碎片吞噬功能。这些技术在不需要重要的专用设备或材料的情况下产生可靠的可量化结果, 但可以很容易地定制以满足研究人员的需要。

BMDMs 的 CFSE 标记髓鞘碎片的治疗应产生清晰的内部化 (图 2)。虽然3小时的互动时间是足够的 BMDMs, 以吞噬足够的补充髓鞘碎片, 为强健的下游检测, 细胞内积累可以观察到, 只需1小时的互动。然而, 一些髓鞘碎片可能仍然存在于细胞表面洗涤后。这可能是由于洗涤不足, 或在吞噬的早期阶段没有完全内化的微粒。
虽然 BMDMs 可以迅速地将髓鞘碎片化, 但在破产可脂滴形成之前需要溶酶体的处理。为了证明, BMDMs 在进行固定和染色之前, 用髓鞘碎片孵育了90分钟, 洗涤和培养了更多的时间 (图 3)。图 4显示了治疗起始和中性脂质出现之间的延迟。还应注意到, 在培养期间, 脂质保持不稳定。BMDMs 将开始代谢积累的脂质不久后滴形成。因此, 我们建议在洗掉 non-engulfed 髓鞘碎片和固定之间不超过24小时。
BMDMs 的定量化表明了该区髓鞘脂代谢率 (图 5)。在90分钟的互动期之后, 细胞被重新 CMCM。在新鲜培养基的早期追逐期间, BMDMs 代谢的吞噬髓鞘碎片脂质成分转化为中性的可血脂。在交互作用后的几个小时内, 破产人积极区域的稳定增长是典型的。然而, 24 小时后, BMDMs 将有 effluxed 或代谢相当一部分的细胞内脂质。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56322/56322fig1.jpg"> 
图 1: 具有代表性的 BMDM 区域性.最初观察到的黏附细胞很少。在3天, 任何换药单元都将被移除。7天, 成熟的骨髓衍生巨噬细胞被观察。缩放 = 50 µm.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56322/56322fig1large.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/56322/56322fig2.jpg"> 
图 2: BMDM 髓鞘碎片吞噬试验的代表性图像.1毫克/毫升 CFSE 标记髓鞘碎片, 在洗涤和固定4% 粉煤灰之前, 细胞被治疗1小时。内化髓鞘碎片可以使用标准的 GFP 过滤装置在 epi 荧光显微镜下进行可视化。缩放 = 50 µm.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56322/56322fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56322/56322fig3.jpg"> 
图 3: 油红色图 (破产) 试验设计.BMDM 治疗1毫克/毫升髓鞘碎片 (1:100 稀释) 90 分钟, 其次是洗涤和文化在新鲜的媒体前固定。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56322/56322fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56322/56322fig4.jpg"> 
图 4: BMDM 在髓鞘碎片吞噬后的染色.在被指定的时点4% 粉煤灰固定后, BMDMs 与破产人和赫斯特33258染色。显示每个时点的代表性图像。缩放 = 100 µm.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56322/56322fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56322/56322fig5.jpg"> 
图 5: 对破产人染色的定量图像分析.为了定量化破产人的染色, 3 口井在20X 成像, 每井5幅图像。所有的图像采集设置都是相同的。误差线 = SEM (n=3)。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56322/56322fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about In Vitro Phagocytosis of Myelin Debris by Bone Marrow-Derived Macrophages at JoVE.com

In Vitro Phagocytosis of Myelin Debris by Bone Marrow-Derived Macrophages

本文采用荧光标记髓鞘碎片和胞内脂滴染色的方法, 对小鼠骨髓源性巨噬细胞的吞噬能力进行评估。

体外骨髓源性巨噬细胞对髓鞘碎片的吞噬

Bone marrow-derived macrophages (BMDMs) are mature leukocytes that serve a critical physiological role as professional phagocytes capable of clearing a ...

in-vitro-phagocytosis-myelin-debris-bone-marrow-derived

Nanyang Technological University

Research

JoVE Journal

Neuroscience

In Vitro Phagocytosis of Myelin Debris by Bone Marrow-Derived Macrophages

12.6K 次观看.  Florida State University. 本文采用荧光标记髓鞘碎片和胞内脂滴染色的方法, 对小鼠骨髓源性巨噬细胞的吞噬能力进行评估。

视频: In Vitro Phagocytosis of Myelin Debris by Bone Marrow-Derived Macrophages

Depletion and Reconstitution of Macrophages in Mice

Macrophages are critical players in the innate immune response to infectious challenge or injury, initiating the innate immune response and directing the acquired immune response. Macrophage dysfunction can lead to an inability to mount an appropriate immune response and as such, has been implicated in many disease processes, including inflammatory bowel diseases. Macrophages display polarized phenotypes that are broadly divided into two categories. Classically activated macrophages, activated by stimulation with IFN&gamma; or LPS, play an essential role in response to bacterial challenge whereas alternatively activated macrophages, activated by IL-4 or IL-13, participate in debris scavenging and tissue remodeling and have been implicated in the resolution phase of inflammation. During an inflammatory response in vivo, macrophages are found amid a complex mixture of infiltrating immune cells and may participate by exacerbating or resolving inflammation. To define the role of macrophages in situ in a whole animal model, it is necessary to examine the effect of depleting macrophages from the complex environment. To ask questions about the role of macrophage phenotype in situ, phenotypically defined polarized macrophages can be derived ex vivo, from bone marrow aspirates and added back to mice, with or without prior depletion of macrophages. In the protocol presented here clodronate-containing liposomes, versus PBS injected controls, were used to deplete colonic macrophages during dextran sodium sulfate (DSS)-induced colitis in mice. In addition, polarized macrophages were derived ex vivo and transferred to mice by intravenous injection. A caveat to this approach is that clodronate-containing liposomes deplete all professional phagocytes, including both dendritic cells and macrophages so to ensure the effect observed by depletion is macrophage-specific, reconstitution of phenotype by adoptive transfer of macrophages is necessary. Systemic macrophage depletion in mice can also be achieved by backcrossing mice onto a CD11b-DTR background, which is an excellent complementary approach. The advantage of clodronate-containing liposome-mediated depletion is that it does not require the time and expense involved in backcrossing mice and it can be used in mice regardless of the background of the mice (C57BL/6, BALB/c, or mixed background).

Macrophages play a central role in homeostasis and pathology in many tissues. The protocol presented here describes methods for depleting macrophages in vivo, deriving polarized macrophages from bone marrow aspirates, and adoptively transferring macrophages into mice. These techniques allow determination of the role that polarized macrophages play in health and disease.

在小鼠体内的巨噬细胞的枯竭和重建

Macrophages are critical players in the innate  immune response to infectious challenge or injury, initiating the innate immune  response and directing ...

科研

免疫与感染

Bone Marrow-derived Macrophage Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.

Macrophages have long been recognized as a critical component of the innate and adaptive immune responses. The recent explosion of knowledge pertaining to evolutionary, genetic, and biochemical aspects of the interaction between macrophages and microbes has renewed scientific attention to macrophages. This article describes a method to differentiate macrophages from mouse bone marrow.

骨髓衍生的巨噬细胞产

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

Study of Phagolysosome Biogenesis in Live Macrophages

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome maturation is the complex and tightly regulated process during which a nascent phagosome undergoes drastic transformation through well-orchestrated interactions with various cellular organelles and compartments in the cytoplasm. This process, which is essential for the physiological function of phagocytic cells by endowing phagosomes with their lytic and bactericidal properties, culminates in fusion of phagosomes with lysosomes and biogenesis of phagolysosomes which is considered to be the last and critical stage of maturation for phagosomes. In this report, we describe a live cell imaging based method for qualitative and quantitative analysis of the dynamic process of lysosome to phagosome content delivery, which is a hallmark of phagolysosome biogenesis. This approach uses IgG-coated microbeads as a model for phagocytosis and fluorophore-conjugated dextran molecules as a luminal lysosomal cargo probe, in order to follow the dynamic delivery of lysosmal content to the phagosomes in real time in live macrophages using time-lapse imaging and confocal laser scanning microscopy. Here we describe in detail the background, the preparation steps and the step-by-step experimental setup to enable easy and precise deployment of this method in other labs. Our described method is simple, robust, and most importantly, can be easily adapted to study phagosomal interactions and maturation in different systems and under various experimental settings such as use of various phagocytic cells types, loss-of-function experiments, different probes, and phagocytic particles.

在现场研究巨噬细胞吞噬溶酶体生源

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome ...

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

Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or M(LPS) macrophages are pro-inflammatory, IL-4 induces alternative macrophage activation and these so-called M2 or M(IL-4) support resolution of inflammation and wound healing. Recent evidence shows the crucial role of metabolic reprogramming in the regulation of M1 and M2 macrophage polarization. 
In this manuscript, an extracellular flux analyzer is applied to assess the metabolic characteristics of naive, M1 and M2 polarized mouse bone marrow-derived macrophages. This instrument uses pH and oxygen sensors to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), which can be related to glycolytic and mitochondrial oxidative metabolism. As such, both glycolysis and mitochondrial oxidative metabolism can be measured in real-time in one single assay.
Using this technique, we demonstrate here that inflammatory M1 macrophages display enhanced glycolytic metabolism and reduced mitochondrial activity. Conversely, anti-inflammatory M2 macrophages show high mitochondrial oxidative phosphorylation (OXPHOS) and are characterized by an enhanced spare respiratory capacity (SRC). 
The presented functional assay serves as a framework to investigate how particular cytokines, pharmacological compounds, gene knock outs or other interventions affect the macrophage&#8217;s metabolic phenotype and inflammatory status.

Metabolic reprogramming is a characteristic and prerequisite for M1 and M2 macrophage polarization. This manuscript describes an assay for the measurement of fundamental parameters of glycolysis and mitochondrial function in mouse bone marrow-derived macrophages. This tool can be applied to investigate how particular factors affect the macrophage&#8217;s metabolism and phenotype.

偏光M1和M2骨髓来源的巨噬细胞的使用实时外流量分析代谢表征

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or ...

Mesenchymal Stem Cell Regulation of Macrophage Phagocytosis; Quantitation and Imaging

Mesenchymal stem cells (MSC) have traditionally been studied for their regenerative properties, but more recently, their immunoregulatory characteristics have been at the forefront. They interact with and regulate immune cell activity. The focus of this study is the MSC regulation of macrophage phagocytic activity. Macrophage (M&#934;) phagocytosis is an important part of the innate immune system response to infection, and the mechanisms through which MSC modulate this response are under active investigation. Presented here is a method to study M&#934; phagocytosis of non-opsonized zymosan particles conjugated to a pH-sensitive fluorescent molecule while in co-culture with MSC. As phagocytic activity increases and the labeled zymosan particles are enclosed within the acidic environment of the phagolysosome, the fluorescence intensity of the pH-sensitive molecule increases. With the appropriate excitation and emission wavelengths, phagocytic activity is measured using a fluorescent spectrophotometer and kinetic data is presented as changes in relative fluorescent units over a 70 min period. To support this quantitative data, the change in the phagocytic activity is visualized using dynamic imaging. Results using this method demonstrate that when in co-culture, MSC enhance M&#934; phagocytosis of non-opsonized zymosan of both naive and IFN-&#947; treated M&#934;. These data add to the current knowledge of MSC regulation of the innate immune system. This method can be applied in future investigations to fully delineate the underlying cellular and molecular mechanisms.

Presented here is a protocol to quantify and produce dynamic images of mesenchymal stem cell (MSC) mediated regulation of macrophage (M&#934;) phagocytosis of non-opsonized yeast (zymosan) particles that are conjugated to a pH-sensitive fluorescent molecule.

间充质干细胞调节巨噬细胞吞噬作用;定量和成像

Mesenchymal stem cells (MSC) have traditionally been studied for their regenerative properties, but more recently, their immunoregulatory characteristics ...

Flow Cytometry Approach to Characterize Phagocytic Properties of Acutely-Isolated Adult Microglia and Brain Macrophages In Vitro

Microglia and central nervous system (CNS)-infiltrating macrophages, collectively called CNS mononuclear phagocytes (CNS-MPs), play central roles in neurological diseases including neurodegeneration and stroke. CNS-MPs are involved in phagocytic clearance of pathological proteins, debris and neuronal synapses, each with distinct underlying molecular pathways. Characterizing these phagocytic properties can provide a functional readout that compliments molecular profiling of microglia using traditional flow cytometry, transcriptomics and proteomics approaches. Phagocytic profiling of microglia has relied on microscopic visualization and in vitro cultures of mouse neonatal microglia. The former approach suffers from limited sampling while the latter approach is inherently poorly reflective of the true in vivo state of adult CNS-MPs. This paper describes optimized protocols to phenotype phagocytic properties of acutely-isolated mouse CNS-MPs by flow cytometry. CNS-MPs are acutely isolated from adult mouse brain using mechanical dissociation followed by density gradient centrifugation, incubated with fluorescent microspheres or fluorescent A&#946; fibrils, washed, and then labeled with panels of antibodies against surface markers (CD11b, CD45). Using this approach, it is possible to compare phagocytic properties of brain-resident microglia with CNS-infiltrating macrophages and then assess the effect of aging and disease pathology on these phagocytic phenotypes. This rapid method also holds potential to functionally phenotype acutely-isolated human CNS-MPs from post-mortem or surgical brain specimens. Additionally, specific mechanisms of phagocytosis by CNS-MP subsets can be investigated by inhibiting select phagocytic pathways.

Assessment of phagocytic properties of microglia and brain macrophages can provide a valuable functional dimension to molecular profiling studies. We describe a validated protocol that uses flow cytometry to rapidly and reliably quantify phagocytosis of fluorescent microspheres and amyloid beta fibrils by acutely-isolated microglia and brain macrophages from mouse models.

Microglia and central nervous system (CNS)-infiltrating macrophages, collectively called CNS mononuclear phagocytes (CNS-MPs), play central roles in ...

Isolation and Culture of Bone Marrow-Derived Macrophages from Mice

Macrophages have important effector functions in homeostasis and inflammation. These cells are present in every tissue in the body and have the important ability to change their profile according to the stimuli present in the microenvironment. Cytokines can profoundly affect macrophage physiology, especially IFN-&#947; and interleukin 4, generating M1 and M2 types respectively. Because of the versatility of these cells, the production of a population of bone marrow-derived macrophages can be a basic step in many experimental models of cell biology. The aim of this protocol is to help researchers in the isolation and culture of macrophages derived from bone marrow progenitors. Bone marrow progenitors from pathogen-free C57BL/6 mice are transformed into macrophages upon exposure to macrophage colony-stimulating factor (M-CSF) that, in this protocol, is obtained from the supernatant of the murine fibroblast lineage L-929. After incubation, mature macrophages are available for use from the 7th to the 10th day. A single animal can be the source of approximately 2 x 107 macrophages. Therefore, it is an ideal protocol for obtaining large amounts of primary macrophages using basic methods of cell culture.

The present protocol describes the isolation and culture of bone marrow-derived macrophages from mice.

从小鼠中分离和培养骨髓来源的巨噬细胞

Macrophages have important effector functions in homeostasis and inflammation. These cells are present in every tissue in the body and have the important ...

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. ...

新生儿骨髓来源巨噬细胞的吞噬试验评价 

Neonatal Mouse Bone Marrow Isolation and Preparation of Bone Marrow-Derived Macrophages

Various techniques for isolating bone marrow from adult mice have been well established. However, isolating bone marrow from neonatal mice is challenging and time-consuming, yet for some models, it is translationally relevant and necessary. This protocol describes an efficient and straightforward method for preparing bone marrow cells from 7-9-day-old pups. These cells can then be further isolated or differentiated into specific cell types of interest. Macrophages are crucial immune cells that play a major role in inflammation and infection. During development, neonatal macrophages contribute significantly to tissue remodeling. Moreover, the phenotype and functions of neonatal macrophages differ from those of their adult counterparts. This protocol also outlines the differentiation of neonatal macrophages from the isolated bone marrow cells in the presence of L929-conditioned medium. Surface markers for differentiated neonatal macrophages were assessed using flow cytometric analysis. To demonstrate functionality, the phagocytic efficiency was also tested using pH-sensitive dye-conjugated Escherichia coli.

作者聚焦：开发一种从新生小鼠中分离骨髓来源的巨噬细胞的可重复方法，以促进早期免疫反应

This protocol describes a non-enzymatic and straightforward method for isolating 7-9-day-old neonatal mouse bone marrow cells and generating differentiated macrophages using a supernatant of L929 cells as a source of granulocyte colony-stimulating factor (M-CSF). The bone marrow-derived macrophages were further analyzed for surface antigens F4/80, CD206, CD11b, and functional competency.

新生小鼠骨髓分离和骨髓来源巨噬细胞的制备

Various techniques for isolating bone marrow from adult mice have been well established. However, isolating bone marrow from neonatal mice is challenging ...