作者感谢妍珠荣格的实验支持, 在体纯化和 Jungjoo 公园的图像突与 PS 曝光。此外, 我们亦感谢锺众的实验室成员进行有益的讨论。这项工作得到了韩国国家研究基金会 (NRF) 资助的韩国政府 (MSIP) (NRF-2016M3C7A1905391 和 NRF-2016R1C1B3006969) (W。C)。

这里描述的所有方法都得到了韩国科学和技术研究所动物护理和使用委员会 (IACUC) 的批准, KA2016-08。
1. 体净化
注意: 这些过程从以前发布的纸张19中进行了修改, 并进行了一些改进, 以提高纯化突的收率 (图 1)。
<ol><li>准备不连续密度梯度介质。<ol>
<li>将以下内容放在冰上: 梯度缓冲液中的3%、10% 和23% 密度梯度解 (以109.54 克蔗糖、606毫克的三、5毫升的0.2 米 EDTA、pH 7.4) 带到250毫升的水;均质缓冲器 (加25毫升的4x 梯度缓冲液和500µL 50 mM 的数码化成74.5 毫升的水);匀浆砂浆和杵。 注意: 在表 1中介绍了密度渐变解决方案和准备工作。要准备3%、10% 和23% 密度梯度解, 请使用原始密度梯度解。</li>
		<li>每三只成年老鼠准备六超清晰离心管。</li>
		<li>在每个离心管的底部加3毫升23% 密度梯度溶液, 用1毫升吸管。然后, 在23% 密度梯度溶液的顶部, 用牧场吸管和1毫升吸管, 慢慢地使一层3毫升的10% 密度梯度溶液。最后, 在顶部添加 3 mL 3% 密度梯度溶液。在此步骤中, 请谨慎避免引入气泡。</li>
		<li>用塑料包盖离心管, 把管子放在冰上。</li>
	</ol>
</li>
	<li>冷高速离心机和 ultracentrifuge 到4° c。</li>
	<li>用70% 乙醇消毒手术设备 (操作剪刀、Castroviejo 弹簧剪刀和弯曲钳)。在冰上准备一个25毫升均质缓冲的小玻璃烧杯, 称量烧杯。</li>
	<li>从三成人 (约12周大) 雄性小鼠身上提取大脑。<ol>
<li>脱臼颈椎, 用手术剪刀割断颈部。用剪刀和弯钳将头部和头骨的皮肤沿中线剥离。用 Castroviejo 弹簧剪刀将嗅球和小脑切除。</li>
	</ol>
</li>
	<li>用弯曲的钳子把剩下的大脑放进烧杯里, 然后称量大脑。用冰冷的均质缓冲液冲洗大脑几次, 以除去大脑表面的血液。 注意: 以前, 推荐9毫升的均质缓冲液到1克脑组织。</li>
	<li>在均质砂浆中加入4毫升的均质缓冲液和1克脑组织。在均匀化大脑的同时, 增加8毫升的均匀化缓冲。</li>
	<li>将匀浆分成两个50毫升的高速离心管。用1毫升的新鲜均质缓冲液清洗砂浆, 并将其添加到管中。 警告: 请尽快执行步骤 1.3-1.7。建议少于20分钟。</li>
	<li>离心机匀在高速离心管在 1000 x g 为10分钟在4° c 以减速的闸。 注: 当减速率为九时, 将减速速率 (制动器) 设置为2或三。</li>
	<li>小心地将上清液添加到新的50毫升管中, 并加到12毫升的均质缓冲器。</li>
	<li>小心缓慢地将2毫升稀释上清液移到3% 密度梯度溶液中, 用1毫升吸管将管子放在冰上。</li>
	<li>离心机在 3.1万 x g 为5分钟在4° c 没有刹车。 注: 设置减速率为 1 (零) 或海岸。</li>
	<li>把管子放在冰上, 在23% 密度梯度溶液和10% 密度梯度溶液之间收集体分数, 用2毫升吸管插入50毫升管。加入冰冷蔗糖/EDTA 缓冲80毫升, 并将其分为四50毫升高速离心管。 注意: 有关带有标记渐变的分馏突, 请参见图 3 。</li>
	<li>离心机在 2万 x g 为30分钟在4˚C 以较少断裂。 注: 当减速的最大速率为九时, 设置减速速率为2或三。</li>
	<li>小心地用1毫升吸管移除上清液重体颗粒与1毫升的等渗生理缓冲 (140 毫米氯化钠, 3 毫米氯化钾, 1.2 毫米氯化镁2, 1.2 mm NaH2PO4, 10 mm HEPES, 和 10 mm 葡萄糖, pH 7.4)20。在等渗生理缓冲中加入1毫升10% 亚砜 (最后浓度为 5%), 并将突收集到一个50毫升的高速离心管中。</li>
	<li>分1毫升成1.5 毫升的管子。用布拉德福法测定突的蛋白质浓度。</li>
	<li>迅速冻结管和存储在一个-80 ° c 深冰箱的管, 直到 pH 值指标共轭。</li>
</ol>2. pH 值指示器共轭
<ol><li>离心机1.5 毫升管与突在 21092 x g 为3-4 分钟在4° c。</li>
	<li>移除上清, 添加200µL 0.1 M Na2CO3 , 并由移混合均匀。 注: Na2CO3被添加到提高 ph 值, 作为琥珀酰亚胺酯最有效地与初级氨基的反应, 在略微碱性的 ph 值。</li>
	<li>增加2µL 的 pH 值指示管和轻轻涡。 注意: 使用1µL 的 pH 值指示每0.3 毫克的体溶液。</li>
	<li>通过用铝箔盖住灯管, 在室温 (RT) 的温度下孵育1-2 小时, 以 30-40 rpm 的温和搅拌, 最大限度地减少光照射。</li>
	<li>停止搅拌, 加入1毫升的 DPBS。</li>
	<li>离心管在 21092 x g 为1-2 分钟。</li>
	<li>移除上清液, 加入1毫升的 DPBS 重, 用温和的移。</li>
	<li>重复步骤 2.6-2.7 至少七次, 以删除未绑定的 pH 值指示器。</li>
	<li>去除上清和重200µL 的 DPBS 与5% 亚甲基亚砜的颗粒。 注意: 在这一点上, 所有的突都是非功能的。我们已经证实, 磷脂 (PS) 已经暴露在突的外层膜上, 它作为 "吃我" 信号 (图 4) 起作用。</li>
</ol>3. 星形胶质细胞
注意: 此协议的星形胶质细胞纯化是根据以前发表的论文21改编的。在本协议中, 对大鼠星形胶质细胞和小胶质的纯化进行了描述。在注释中还描述了纯化小鼠星形胶质细胞的具体步骤。
<ol><li>净化前一天
	<ol>
<li>准备预涂的细胞培养皿和溶液。</li>
		<li>准备五145毫米 x 20 mm 的培养皿, 25 毫升的50毫米三盐酸 (pH 9.5)。在培养皿中分别放置初级或二次抗体治疗, 如下所述。一夜之间在冰箱里孵育盘子。 BSL-1 盘:20 µL 5 毫克/毫升 BSL-1 (加纳单叶凝集素) 二次唯一盘:60 µL 2.4 毫克/毫升山羊抗鼠 IgG + IgM (H + L) CD45 盘:60 µL 2.4 毫克/毫升山羊抗鼠 IgG + IgM (H + L) O4 盘:60 µL 2.4 毫克/毫升山羊抗鼠 IgM (µ链特异性) Itgb5 (整合素β5) 大鼠星形胶质细胞:60 µL 2.4 毫克/毫升山羊抗鼠 IgG + IgM (H + L) 注意: 要在培养皿的疏水性表面附加二次抗体, 建议在低温下缓慢附着。但是, 在37° c 的情况下, 有可能在2小时的第二抗体上涂上培养皿。用于星形胶质细胞的抗体根据不同的动物种类而有所不同;例如, 小鼠星形胶质细胞 HepaCAM 盘:60 µL 2.4 毫克/毫升山羊抗鼠 IgG + IgM (H + L)</li>
	</ol></li>
	<li>净化日
	<ol>
<li>准备 immunopanning</li>
		<li>准备50毫升的管子, 并在管子上添加存货。详细的库存解决方案准备工作在表 1中提供。<ol>
<li>制备管 1 (酶):22 毫升的酶库存 (酶库存: 1x 厄尔的平衡盐溶液 (EBSS) + 0.46% D (+)-葡萄糖 + 26 mm NaHCO3和0.5 毫米 EDTA)
</li>
			<li>准备管 2 (低蛋):42 毫升抑制剂库存 (抑制剂库存: 1x EBSS + 0.46% D (+)-葡萄糖 + 26 Mm NaHCO3)。</li>
			<li>准备管 3 (高蛋):10 毫升抑制剂库存。</li>
		</ol>
</li>
		<li>将0.22 µm 注射器过滤器连接到 CO2 (95% O2和 5% co2) 槽的 2 mL 管的提示。气泡 CO2成管1-3。当解决方案变为橙色时停止冒泡。</li>
		<li>完成解决方案。 警告: 在使用酶激活之前, 在34° c 的试管1中完成并孵育溶液15分钟。<ol>
<li>制备管 1 (酶):22 毫升的酶库存 + 180 U 的木瓜蛋白酶 + 0.004 克-l-半胱氨酸。</li>
			<li>准备管 2 (低蛋):42 毫升抑制剂库存 + 3 毫升低蛋 + 200 µL dnasei。</li>
			<li>准备管 3 (高蛋):10 毫升抑制剂库存 + 2 毫升高蛋 + 20 µL dnasei。</li>
			<li>准备管 4 (0.2% bsa):19 毫升的 1X DPBS + 1 毫升 4% bsa + 8 µL dnasei。</li>
			<li>准备管 5 (0.02% BSA):45 毫升的 1X DPBS + 5 毫升的管 4 + 50 µL dnasei。</li>
		</ol>
</li>
		<li>在34° c 预热水浴和热块。</li>
	</ol></li>
	<li>提取大鼠皮层。<ol>
<li>用70% 乙醇消毒手术设备, 用 DPBS 准备100毫米的培养皿。</li>
		<li>准备一个亚克力盒子。在盒子的底部放置两到三层的组织, 并在盒子里加入1毫升的异氟醚。</li>
		<li>麻醉的小狗在箱子里20-40 秒, 用钳子麻醉脚捏。使用带有 DPBS 的10毫升注射器将心脏22和灌注的幼崽暴露。 注: 在小胶质细胞平移的过程中, 灌流是用来避免血液污染的。</li>
		<li>用剪刀切开小狗颈上颈部线, 沿中线切割头部皮肤。沿中线切开颅骨, 用弯曲钳打开颅骨。</li>
		<li>用 Castroviejo 弹簧剪刀切除小脑。取出剩下的大脑, 放在一个100毫米的培养皿里, 里面装满了 DPBS。</li>
		<li>在显微镜下用 Castroviejo 弹簧剪刀从皮层中取出其他区域, 如中脑和海马。用细钳取出脑膜。</li>
		<li>将大脑皮层移植到一个新的60毫米的盘子里, 然后把剩下的 DPBS 放在盘子里。用 No. 10 刀片切开组织。</li>
	</ol></li>
	<li>将皮质游离成单个细胞悬浮<ol>
<li>将过滤管1溶液倒入60毫米的盘中, 加入50µL 的 DNase1。在盘子里旋转溶液。 注: dnasei 用于抑制 DNA 从破裂细胞中聚集。</li>
		<li>把盘子放进一个34° c 的热块中, 盖上盖子, 中间有个洞。使用烙铁使60毫米培养皿的盖子上的孔。</li>
		<li>将22µm 注射器过滤器连接至 CO2罐管, 并将过滤器尖放入孔中。</li>
		<li>在34° c 下孵育45分钟, 每10至15分钟将溶液旋流在盘中。</li>
	</ol></li>
	<li>完成准备的平移菜。<ol>
<li>把从冰箱里涂上二次抗体的平移碟提前一天移走。在 RT 中留下 BSL-1 盘和二次抗体仅盘。</li>
		<li>用20毫升的 DPBS 洗其他菜三次。</li>
		<li>在盘子里倒入足够数量的 0.02% BSA。将特定的初级抗体放入相应的菜肴中: CD45 盘:12 毫升 0.02% BSA + 20 µL 0.5 毫克/毫升大鼠抗鼠 CD45 Itgb5 盘:12 毫升 0.02% BSA + 20 µL 0.5 毫克/毫升 Itgb5 O4 盘: 8 毫升 0.02% BSA 和4毫升 O4 抗体 注意: 对于小鼠 immunopanning, 使用小鼠 CD45 (0.5 毫克/毫升) 对星形胶质细胞和 HepaCAM (0.5 毫克/毫升) 的实验。</li>
		<li>准备30毫升的 30% FBS 与 neurobasal 媒体和10毫升的 1X EBSS。加热两个解决方案在一个34° c 水浴。</li>
	</ol></li>
	<li>抑制木瓜蛋白酶的反应。<ol>
<li>将木瓜蛋白酶处理的组织转移到一个新的50毫升管。等待组织的解决。吸入液体。</li>
		<li>加入4毫升的低蛋溶液到管和漩涡的解决方案, 以清洗细胞。</li>
		<li>重复步骤 3.6. 1-3. 6.2 三次停止酶反应。</li>
	</ol></li>
	<li>研制皮层组织。<ol>
<li>在浴缸中加入6毫升的低蛋。用5毫升吸管快速吸气和释放组织。 注意: 不要引入气泡。</li>
		<li>准备一个新的50毫升管和增加5毫升的低蛋。在一个新的试管里收集单个细胞。</li>
		<li>重复步骤 3.6. 1-3. 6.2 两次, 并将5毫升吸管改为1毫升吸管。</li>
		<li>重复研磨, 直到超过95% 的组织是不可见的。</li>
		<li>使一层高蛋溶液从管3与10毫升的吸管低于低蛋含有离解细胞。</li>
		<li>离心机在 190 x g 为6分钟以少量闸。 注: 当减速的最大速度为九时, 设置减速率为一两个。</li>
	</ol></li>
	<li>过滤单个细胞。<ol>
<li>小心地吸取上清液的吸力, 然后重3毫升的 0.02% BSA 溶液与1毫升吸管的颗粒。</li>
		<li>添加 0.02% BSA 解决方案高达9毫升。</li>
		<li>准备一个新的50毫升管和20µm 细胞过滤器的顶部的管。用1毫升 0.02% BSA 溶液润湿细胞过滤器。</li>
		<li>将单细胞悬浮液转移到细胞过滤器。一次过滤1毫升。</li>
		<li>用3毫升 0.02% BSA 溶液清洗细胞过滤器。不要使超过15毫升的过滤单细胞悬浮。</li>
		<li>在34° c 水浴中孵育45-60 分钟, 用于细胞恢复。细胞恢复步骤允许细胞表面蛋白孵育膜。</li>
	</ol></li>
	<li>执行单元格平移。<ol>
<li>用20毫升的 DPBS 洗 CD45 平移盘三次。使用前立即用20毫升的 DPBS 冲洗每道菜三次。</li>
		<li>将单细胞悬浮液倒入 CD45 平移盘中。把盘子放28分钟, 摇动盘子14分钟。要从底部移除未绑定的单元格, 请小心地摇动盘子。</li>
		<li>将电池悬浮液转移到二次唯一的盘中。等待20分钟, 仔细地摇动盘子10分钟。要从 CD45 盘中获取小胶质细胞, 请到步骤3.9.2。</li>
		<li>将细胞悬浮液转移到 BSL-1 盘中。把盘子放10到12分钟。</li>
		<li>转入 O4 盘。等待20分钟, 摇动10分钟。</li>
		<li>转入 Itgb5 盘。把盘子放40分钟, 轻轻摇动每10分钟。 注: 对于鼠标平移, 将电池悬浮液转移到 HepaCAM 盘中。</li>
	</ol></li>
	<li>从盘子里分离出细胞。<ol>
<li>混合400µL 的胰蛋白酶 (3万 U/毫升在 EBSS) 与 preincubated 8 毫升的 1X EBSS。</li>
		<li>把分离的细胞悬浮在盘子里倒入一个废物桶。</li>
		<li>用 DPBS 洗碗八次。</li>
		<li>将8毫升的 EBSS 与胰蛋白酶倒入盘中。</li>
		<li>在37° c 孵化箱中孵育5至10分钟的盘。孵育10分钟的 CD45 菜和5分钟的 Itgb5 和 HepaCAM 菜。</li>
		<li>在显微镜下点击盘子并观察盘子。 注意: 如果大多数的星形胶质细胞没有分离, 就把盘子放回孵卵器里再放5分钟。</li>
		<li>将10毫升的 30% FBS 倒入盘中, 以中和胰蛋白酶。</li>
		<li>在整个盘子里向上和向下移去分离星形胶质细胞或小胶质。</li>
		<li>将溶液转化为50毫升的管子。</li>
		<li>加入10毫升的 30% FBS 和200µL 的 dnasei 到菜。再次分离单元格。dnasei 用于抑制 DNA 从破裂细胞中聚集。</li>
		<li>把溶液转到管子里。如果细胞留在盘子里, 再加10毫升的 30% FBS, 然后再分离细胞。</li>
	</ol></li>
	<li>板细胞。<ol>
<li>离心管在 213 x g 为10分钟。</li>
		<li>用培养基吸上清液, 重细胞颗粒。 注: 使用 immunopanned 星形胶质细胞基本培养基 (ip-ABM), 为胶质和视网膜小胶质碱基介质 (MBM) 与 immunopanned 胶质组织的多孔材料-条件介质 (ip-ACM)。在材料目录中介绍了 IP-ABM 和 MBM 组合。</li>
		<li>用例计数细胞。</li>
		<li>将星形胶质细胞和小胶质板分别印在 PDL 涂层板上。 注: 在使用前准备 PDL 涂层板。要准备 PDL 涂层板, 在5毫升蒸馏水中加入50µL 1 毫克/毫升的聚-d-赖氨酸, 并将适量的溶液倒入井板/盘中, 等待30分钟. 用蒸馏水冲洗盘子/盘子三次。</li>
		<li>孵化板。改变一半的媒体每7天的星形胶质细胞和3天的小胶质。</li>
	</ol></li>
</ol>4. 收集 IP-ACM
<ol><li>当星形胶质细胞过度汇合时, 准备一个100毫米的盘子。</li>
	<li>丢弃介质, 用10毫升的 DPBS 洗碗三次。</li>
	<li>将15毫升的低蛋白培养基倒入盘中。 注: 低蛋白质介质成分在材料表中呈现。</li>
	<li>在37° c 孵化皿中孵育7至10天。</li>
	<li>收集和集中的介质与 10k (或 30k) 离心过滤管。</li>
	<li>离心管在 851 x g 在4° c。 注: 离心管, 直到剩余的介质小于1毫升。</li>
	<li>收集集中媒体 (IP-ACM) 到一个新的1.5 毫升管。</li>
	<li>用定量分析法测量 IP-ACM 的浓度</li>
</ol>5. 吞噬活成像检测 (图 2)
<ol><li>准备一个24井板 (或任何板/碟, 准备活成像), 其中星形胶质细胞是汇合 (一般, 7 至10天的孵化后, 净化是理想的稳定细胞)。</li>
	<li>取出每口井中的介质, 用1毫升的 DPBS 洗净, 三次。为了尽量减少空气暴露, 迅速冲洗水井。</li>
	<li>添加300µL 的 ip-ABM 与5µL pH 指标-共轭突和其他因素, 如 ip-ACM, 可以调节胶质细胞吞噬功能。</li>
	<li>在 CO2孵化器中孵育板40分钟。这一步允许 pH 指示-共轭突沉淀到底部的板块。</li>
	<li>用未粘合的 pH 指示物去除介质-共轭突和洗涤每井与1毫升 DPBS, 三次。 注意: 不要洗得太严厉。为了尽量减少空气暴露, 迅速冲洗水井。</li>
	<li>添加500µL 的 IP-ABM 与其他因素, 以测试入井。</li>
	<li>把盘子放到活成像仪器上, 选择位置。调整焦点、曝光时间、亮度和 LED 电源。 注意: 曝光时间、亮度和 LED 功率的设定是根据荧光强度和实验目的而变化的。在活成像分析与 pH 指示-共轭突, 我们通常设置这些值如下: 曝光: 〜150-200 毫秒, 亮度:15, 和 LED 功率: 4-6。</li>
	<li>设置图像格式、时间间隔和实时成像的总周期数。根据选择的职位数, 将时间间隔设置为1或2小时。2小时间隔的建议时, 超过150职位被选中的实时成像。</li>
	<li>开始现场成像实验</li>
	<li>分析数据。<ol>
<li>打开斐济计划。</li>
		<li>导入图像序列。将图像转换为8位灰度。</li>
		<li>执行背景减法, 滚动条半径为50像素。</li>
		<li>启动时间序列分析器 V3 插件。 注意: 时间序列分析器 V3 插件的地址显示在材料表中。</li>
		<li>拖动图像中的感兴趣区域 (roi), 然后在 roi 管理器中单击 "添加"。</li>
		<li>在时间序列 V3_0 中单击 "获得总强度"。</li>
		<li>保存结果并集成数据。</li>
	</ol>
</li>
</ol>

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作者没有什么可透露的。

在这篇文章中, 我们提出了一种长期活体成像体外的方法, 利用纯化的胶质细胞和 pH 指标-共轭突的吞噬试验。我们发现, 与小胶质细胞相比, 星形胶质在吞噬突时具有不同的吞没和降解能力。此外, 我们的数据表明, 星形胶质细胞分泌的因素, 其中包含桥接分子, 如 GAS6, 蛋白质, 和 MEGE8, 是必不可少的有效的 PS 依赖的吞噬神经细胞的大脑。此外, Megf10高星形胶质细胞显示有缺陷的吞噬突?...

胶质细胞是中枢神经系统中主要的细胞类型, 它指的是大脑中不可兴奋的细胞。以前, 胶质细胞被认为是单纯的支持细胞, 主要在维持神经元存活和基底突触特性方面扮演被动角色。然而, 新出现的证据表明, 胶质细胞在神经生物学的各个方面发挥更积极的作用, 如维持脑内稳态, 调停突触形成1,2,3和突触消除4、5和调制突触可塑性67。中枢神经系统中的胶质细胞包括星形胶质, 小胶质, 和突。在这些细胞中, 胶质细胞和小胶质蛋白通过吞噬突触来发挥吞噬作用4,5, 凋亡细胞8, 神经碎片9, 以及致病性蛋白质, 如淀粉样β斑块10,11。在发育中的大脑中, 星形胶质细胞通过 MERTK 和 MEGF10-dependent 吞噬功能消除了背侧膝状核 (dLGN) 的突触 (4。同样, 小胶质细胞也消除 C1q-coated 突触在发育阶段通过经典补充级联5。有趣的是, 有人认为, 突触修剪的缺陷可能是几种神经紊乱的引发器之一。例如, 它已经表明, 在补充成分 4 (C4) 的突变, 这增加了补充介导的突触修剪由小胶质细胞, 强烈与精神分裂症的患病率在人12。最近的一篇论文还表明, 经典的补体通路是 hyperactivated 在 AD 的起始阶段, 并诱导早期突触损失在这种疾病13。
与小胶质细胞介导的吞噬作用相比, 星形胶质细胞介导的吞噬作用是否有助于各种神经功能紊乱的发生和进展, 尚不清楚。然而, 最近的一篇论文指出, 改变细胞的正常突触修剪率的因素可能会扰乱大脑的稳态, 并导致 AD 易感性和病理14。星形胶质细胞的突触修剪率由载脂蛋白异构体强力控制, 具有 ad 的保护性等位基因 (ApoE2), 大大提高 ad (ApoE4) 的速率和风险等位基因, 显著降低率。此外, 表达ApoE4的转基因小鼠的突触 C1q 比对照组或ApoE2小鼠14大得多。这些数据表明, 受损的星形胶质细胞介导的吞噬在早期 AD 大脑可能会诱发衰老 C1q-coated 突触/突触碎片的积累, 激活补体介导的胶质吞噬, 驱动突触变性.在ApoE4载体中, 星形胶质细胞的吞噬能力受损也可能导致受影响的大脑中淀粉样蛋白β斑块的失控积累。
此外, 它已经表明, 在老年果蝇大脑中的胶质细胞失去了他们的吞噬能力, 由于减少翻译的德雷柏, 一个同源的Megf10 , 星形细胞用于吞噬突触。恢复德雷柏的水平挽救了胶质细胞的吞噬能力, 它有效地清除受损的大脑中的轴突碎片与年轻的大脑一样的程度, 表明老化诱导的吞噬能力的变化星形胶质细胞可能导致大脑稳态破坏15。
根据这些新发现, 调节星形胶质细胞的吞噬能力可能是预防和治疗各种神经紊乱的一种有吸引力的治疗策略。在这方面, 有几次试图提高星形胶质细胞的吞噬能力, 例如, 通过酸性纳米粒子诱导溶酶体的酸化16和表达转录因子 EB (TFEB), 可以提高溶生物17。尽管有这些尝试, 但目前仍不清楚的是, 星形胶质细胞和胶质在它们的吞噬动力学上有何不同, 我们是否应该增加或减少它们在各种疾病中的吞噬能力。
本文提出了一种新的体外检测星形胶质细胞吞噬能力的方法。数据显示星形胶质细胞和小胶质吞没和降解的不同动力学。星形胶质细胞被条件培养基 (ACM), 其中包含的分泌因素, 从胶质, 是必不可少的有效吞噬的星形胶质细胞和小胶质。此外, Megf10, 在星形胶质细胞的吞噬受体和Ced-1和德雷柏同源, 发挥关键的作用, 在胶质-介导吞噬8,18。

<table>
	<tbody>
		<tr>
			<td>Synaptosome purification</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll</td>
			<td>GE healthcare life sciences</td>
			<td>17-0891-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Quick Start Bradford Protein Assay Kit 2</td>
			<td>BIO-RAD</td>
			<td>5000202</td>
			<td></td>
		</tr>
		<tr>
			<td>pH indicator conjugation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide(DMSO)</td>
			<td>LPS solution</td>
			<td>DMSO100</td>
			<td></td>
		</tr>
		<tr>
			<td>pHrodo red, succinimidyl ester</td>
			<td>Molecula probes</td>
			<td>P36600</td>
			<td></td>
		</tr>
		<tr>
			<td>Immunopanning</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10X Earle&#8217;s balanced salt solution (EBSS)</td>
			<td>Sigma</td>
			<td>E7510</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Bovogen</td>
			<td>BSA025</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxyrebonuclease 1 (DNase)</td>
			<td>Worthington</td>
			<td>Is002007</td>
			<td></td>
		</tr>
		<tr>
			<td>(DMEM)</td>
			<td>Gibco</td>
			<td>11960-044</td>
			<td></td>
		</tr>
		<tr>
			<td>(dPBS)</td>
			<td>Welgene</td>
			<td>LB001-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Gibco</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Griffonia Simplicifolia Lectin(BSL-1)</td>
			<td>Vector Labs</td>
			<td>L-1100</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG+IgM(H+L)</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-005-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgM (&#956;-chain)</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-005-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin-binding epidermal growth factor</td>
			<td>Sigma</td>
			<td>E4643</td>
			<td></td>
		</tr>
		<tr>
			<td>Human HepaCAM antibody</td>
			<td>R&#38;D systems</td>
			<td>MAB4108</td>
			<td></td>
		</tr>
		<tr>
			<td>Integrin beta 5 monoclonal antibody (KN52)</td>
			<td>eBioscience</td>
			<td>14-0497-82</td>
			<td></td>
		</tr>
		<tr>
			<td>L-cysteine</td>
			<td>Sigma</td>
			<td>C7880</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamate</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>N-acetly-L-cyteine (NAC)</td>
			<td>Sigma</td>
			<td>A8199</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal media</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>O4 hybridoma supernatant(mouse IgM)</td>
			<td>Bansal et al.23</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Papain</td>
			<td>Worthington</td>
			<td>Is003126</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluristrainer 20 &#956;m</td>
			<td>PluriSelect</td>
			<td>43-50020-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-lysine</td>
			<td>Sigma</td>
			<td>P6407</td>
			<td></td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma</td>
			<td>P8783</td>
			<td></td>
		</tr>
		<tr>
			<td>Putrescine dihydrochloride</td>
			<td>Sigma</td>
			<td>P5780</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified rat anti-mouse CD45</td>
			<td>BD Pharmingen</td>
			<td>550539</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified mouse anti-rat CD45</td>
			<td>BD Pharmingen</td>
			<td>554875</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Gibco</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium selenite</td>
			<td>Sigma</td>
			<td>S5261</td>
			<td></td>
		</tr>
		<tr>
			<td>Transferrin</td>
			<td>Sigma</td>
			<td>T1147</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma</td>
			<td>T9935</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin inhibitor</td>
			<td>Worthington</td>
			<td>LS003086</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-clear tube (Tube, Thinwall, Ultra-Clear)</td>
			<td>Beckman Coulter</td>
			<td>344059</td>
			<td></td>
		</tr>
		<tr>
			<td>Collect IP-ACM</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Macrosep Advance Centrifugal Devices with Omega Membrane (10k)</td>
			<td>PALL</td>
			<td>MAP010C37</td>
			<td></td>
		</tr>
		<tr>
			<td>Macrosep Advance Centrifugal Devices with Omega Membrane (30k)</td>
			<td>PALL</td>
			<td>MAP030C37</td>
			<td></td>
		</tr>
		<tr>
			<td>Phagocytosis live imaging assay</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Juli stage</td>
			<td>NanoEntek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Time Series Analyzer V3 plugins</td>
			<td>https://imagej.nih.gov/ij/plugins/time-series.html</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a novel in vitro live-imaging assay of astrocyte-mediated phagocytosis using ph indicator-conjugated synaptosomes

星形胶质细胞是大脑中的主要类型, 直接接触突触和血管。虽然胶质细胞已被认为是主要的免疫细胞, 只有吞噬在大脑中, 最近的研究表明, 星形细胞也参与了各种吞噬过程, 如发育突触消除和清除阿尔茨海默病 (AD) 的淀粉样β斑块。尽管有这些发现, 但与小胶质细胞相比, 星形胶质细胞的吞没和靶向降解的效率还不清楚。这种缺乏信息的主要原因是缺乏一个分析系统, 其中星形胶质细胞和小胶质细胞介导的吞噬动力学是很容易比较的。为了实现这一目标, 我们开发了一个长期的活体成像体外吞噬试验, 以评估纯化的星形胶质细胞和小胶质的吞噬能力。在这种测定中, 吞没和降解的实时检测是可能的使用 pH 指示物共轭突, 它发出明亮的红色荧光在酸性细胞器, 如溶酶体。我们的新的检测方法提供了简单和有效的活体成像探测的吞噬。此外, 这种体外吞噬试验可以作为一个筛选平台, 用来识别能增强或抑制星形胶质细胞吞噬能力的化学物质和化合物。由于突触修剪故障和致病蛋白积累已被证明是导致精神疾病或神经退行性疾病, 化学物质和化合物, 调节吞噬能力的胶质细胞应有助于治疗各种神经紊乱

在这体外吞噬试验长期活成像, 我们使用突从成年小鼠脑匀, 这是分离的梯度解决方案之间的23% 梯度溶液和10% 梯度溶液由离心 (图 3)。在制备后, 突暴露 ps 在他们的外膜 (图 4), 表明他们失去了他们的功能, 可以识别的 ps 受体在星形胶质细胞和小胶质。如图 5所示, pH 值指示器-共轭突在被星形胶质细?...

Watch this Scientific Journal Video about A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes at JoVE.com

A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

该协议提供了一个体外活体成像吞噬试验来测量星形胶质细胞的吞噬能力。纯化大鼠星形胶质细胞和小胶质和 pH 指标-共轭突。该方法可检测实时吞没和降解动力学, 为确定影响星形胶质细胞吞噬能力的因素提供了一个合适的筛选平台。

一种新的体外活体成像法测定星形胶质细胞的吞噬作用 pH 指标-共轭突

Astrocytes are the major cell type in the brain and directly contact synapses and blood vessels. Although microglial cells have been considered the major ...

a-novel-vitro-live-imaging-assay-astrocyte-mediated-phagocytosis

Research

JoVE Journal

Neuroscience

A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

12.0K 次观看.  Korea Advanced Institute of Science and Technology. 该协议提供了一个体外活体成像吞噬试验来测量星形胶质细胞的吞噬能力。纯化大鼠星形胶质细胞和小胶质和 pH 指标-共轭突。该方法可检测实时吞没和降解动力学, 为确定影响星形胶质细胞吞噬能力的因素提供了一个合适的筛选平台。

视频: A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and learning and memory. There are four known families of PKC isoforms in vertebrates: classical PKCs (&alpha;, &beta;I, &beta;II and &gamma;), novel type I PKCs (&#949; and &eta;), novel type II PKCs (&delta; and &theta;), and atypical PKCs (&zeta; and &iota;). The classical PKCs are activated by Ca2+ and diacylclycerol (DAG), while the novel PKCs are activated by DAG, but are Ca2+-independent. The atypical PKCs are activated by neither Ca2+ nor DAG. In Aplysia californica, our model system to study memory formation, there are three nervous system specific PKC isoforms one from each major class, namely the conventional PKC Apl I, the novel type I PKC Apl II and the atypical PKC Apl III. PKCs are lipid-activated kinases and thus activation of classical and novel PKCs in response to extracellular signals has been frequently correlated with PKC translocation from the cytoplasm to the plasma membrane. Therefore, visualizing PKC translocation in real time in live cells has become an invaluable tool for elucidating the signal transduction pathways that lead to PKC activation. For instance, this technique has allowed for us to establish that different isoforms of PKC translocate under different conditions to mediate distinct types of synaptic plasticity and that serotonin (5HT) activation of PKC Apl II requires production of both DAG and phosphatidic acid (PA) for translocation 1-2. Importantly, the ability to visualize the same neuron repeatedly has allowed us, for example, to measure desensitization of the PKC response in exquisite detail 3. In this video, we demonstrate each step of preparing Sf9 cell cultures, cultures of Aplysia sensory neurons have been described in another video article 4, expressing fluorescently tagged PKCs in Sf9 cells and in Aplysia sensory neurons and live-imaging of PKC translocation in response to different activators using laser-scanning microscopy.

In this video, we demonstrate visualization of PKC translocation in living cells using fluorescently tagged PKCs.

在Sf9细胞，并在海兔感觉神经元PKC移位实时成像

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and ...

科研

神经科学

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

在发展中国家斑马鱼的前脑的神经祖细胞克隆相关的定时实时成像

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

Live Imaging of Dorsal Root Axons after Rhizotomy

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. Regeneration of dorsal root (DR) axons into spinal cord is prevented at the dorsal root entry zone (DREZ), the interface between the CNS and PNS. Our understanding of the molecular and cellular events that prevent regeneration at DREZ is incomplete, in part because complex changes associated with nerve injury have been deduced from postmortem analyses. Dynamic cellular processes, such as axon regeneration, are best studied with techniques that capture real-time events with multiple observations of each living animal. Our ability to monitor neurons serially in vivo has increased dramatically owing to revolutionary innovations in optics and mouse transgenics. Several lines of thy1-GFP transgenic mice, in which subsets of neurons are genetically labeled in distinct fluorescent colors, permit individual neurons to be imaged in vivo1. These mice have been used extensively for in vivo imaging of muscle2-4 and brain5-7, and have provided novel insights into physiological mechanisms that static analyses could not have resolved. Imaging studies of neurons in living spinal cord have only recently begun. Lichtman and his colleagues first demonstrated their feasibility by tracking injured dorsal column (DC) axons with wide-field microscopy8,9. Multi-photon in vivo imaging of deeply positioned DC axons, microglia and blood vessels has also been accomplished10. Over the last few years, we have pioneered in applying in vivo imaging to monitor regeneration of DR axons using wide-field microscopy and H line of thy1-YFP mice. These studies have led us to a novel hypothesis about why DR axons are prevented from regenerating within the spinal cord11.
In H line of thy1-YFP mice, distinct YFP+ axons are superficially positioned, which allows several axons to be monitored simultaneously. We have learned that DR axons arriving at DREZ are better imaged in lumbar than in cervical spinal cord. In the present report we describe several strategies that we have found useful to assure successful long-term and repeated imaging of regenerating DR axons. These include methods that eliminate repeated intubation and respiratory interruption, minimize surgery-associated stress and scar formation, and acquire stable images at high resolution without phototoxicity.

An in vivo imaging protocol to monitor primary sensory axons following dorsal root crush is described. The procedures utilize wide-field fluorescence microscopy and thy1-YFP transgenic mice, and permit repeated imaging of axon regeneration over 4 cm in the PNS and axon interactions with the interface of the CNS.

实时成像背根轴突后根切断术

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. ...

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of existing mouse lines for genetic cell lineage tracing: a tamoxifen-inducible Cre line and a Cre reporter line expressing dsRed upon Cre-mediated recombination. By using a relatively low level of tamoxifen, we were able to induce recombination in a small number of cells, permitting us to follow individual cell divisions. Additionally, we observed the transcriptional response to Sonic Hedgehog (Shh) signaling using an Olig2-eGFP transgenic line 1-3 and we monitored formation of cilia by infecting the cultured slice with virus expressing the cilia marker, Sstr3-GFP 4. In order to image the neuroepithelium, we harvested embryos at E8.5, isolated the neural tube, mounted the neural slice in proper culturing conditions into the imaging chamber and performed time-lapse confocal imaging. Our ex vivo live imaging method enables us to trace single cell divisions to assess the relative timing of primary cilia formation and Shh response in a physiologically relevant manner. This method can be easily adapted using distinct fluorescent markers and provides the field the tools with which to monitor cell behavior in situ and in real time.

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Here we develop the tools necessary for ex vivo live imaging to trace single cell divisions in the mouse E8.5 neuroepithelium

体外实时成像在小鼠神经上皮单细胞分裂

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of ...

Live Imaging of Drosophila Larval Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given their relevance to development and disease, understanding the mechanisms that govern asymmetric stem cell division has been a robust area of study. Because they are genetically tractable and undergo successive rounds of cell division about once every hour, the stem cells of the Drosophila central nervous system, or neuroblasts, are indispensable models for the study of stem cell division. About 100 neural stem cells are located near the surface of each of the two larval brain lobes, making this model system particularly useful for live imaging microscopy studies. In this work, we review several approaches widely used to visualize stem cell divisions, and we address the relative advantages and disadvantages of those techniques that employ dissociated versus intact brain tissues. We also detail our simplified protocol used to explant whole brains from third instar larvae for live cell imaging and fixed analysis applications.

Live Imaging of Drosophila Larval Neuroblasts

This protocol details a streamlined method used to conduct live cell imaging in the context of an intact larval brain. Live cell imaging approaches are invaluable for the study of asymmetric neural stem cell divisions as well as other neurogenic and developmental processes, consistently uncovering mechanisms that were previously overlooked.

的实时成像<em&gt;果蝇</em&gt;幼虫神经母细胞

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given ...

Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various neurological deficits. The current ex vivo live imaging of motile ependymal cilia technique allows for a detailed study of ciliary dynamics following several steps. These steps include: mice euthanasia with carbon dioxide according to protocols of The University of Toledo&#8217;s Institutional Animal Care and Use Committee (IACUC); craniectomy followed by brain removal and sagittal brain dissection with a vibratome or sharp blade to obtain very thin sections through the brain lateral ventricles, where the ependymal cilia can be visualized. Incubation of the brain&#8217;s slices in a customized glass-bottom plate containing Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)/High-Glucose at 37 &#176;C in the presence of 95%/5% O2/CO2 mixture is essential to keep the tissue alive during the experiment. A video of the cilia beating is then recorded using a high-resolution differential interference contrast microscope. The video is then analyzed frame by frame to calculate the ciliary beating frequency. This allows distinct classification of the ependymal cells into three categories or types based on their ciliary beating frequency and angle. Furthermore, this technique allows the use of high-speed fluorescence imaging analysis to characterize the unique intracellular calcium oscillation properties of ependymal cells as well as the effect of pharmacological agents on the calcium oscillations and the ciliary beating frequency. In addition, this technique is suitable for immunofluorescence imaging for ciliary structure and ciliary protein localization studies. This is particularly important in disease diagnosis and phenotype studies. The main limitation of the technique is attributed to the decrease in live motile cilia movement as the brain tissue starts to die.

Using high-resolution differential interference contrast (DIC) microscopy, an ex vivo observation of the beating of motile ependymal cilia located within the mouse brain ventricles is demonstrated by live-imaging. The technique allows a recording of the unique ciliary beating frequency and beating angle as well as their intracellular calcium oscillation pacing properties.

室管膜纤毛在小鼠脑侧脑室实时成像

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various ...

Novel Assay for Cold Nociception in Drosophila Larvae

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory machinery, such as in patients experiencing peripheral neuropathy or injury-induced sensitization, are not well characterized. The genetically tractable Drosophila model has been used to study the cells and genes required for noxious heat detection, which has yielded multiple conserved genes of interest. Little is known however about the cells and receptors important for noxious cold sensing. Although, Drosophila does not survive prolonged exposure to cold temperatures (&#8804;10 &#186;C), and will avoid cool, preferring warmer temperatures in behavioral preference assays, how they sense and possibly avoid noxious cold stimuli has only recently been investigated.
Here we describe and characterize the first noxious cold (&#8804;10 &#186;C) behavioral assay in Drosophila. Using this tool and assay, we show an investigator how to qualitatively and quantitatively assess cold nociceptive behaviors. This can be done under normal/healthy culture conditions, or presumably in the context of disease, injury or sensitization. Further, this assay can be applied to larvae selected for desired genotypes, which might impact thermosensation, pain, or nociceptive sensitization. Given that pain is a highly conserved process, using this assay to further study&#160;thermal nociception will likely glean important understanding of pain processes in other species, including vertebrates.

Novel Assay for Cold Nociception in Drosophila Larvae

Here we demonstrate a novel assay to study cold nociception in Drosophila larvae. This assay utilizes a custom-built Peltier probe capable of applying a focal noxious cold stimulus and results in quantifiable cold-specific behaviors. This technique will allow further cellular and molecular dissection of cold nociception.

新的分析冷痛觉在<em&gt;果蝇</em&gt;幼虫

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory ...

Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or postmitotic neurons. Later, some progenitors switch to a gliogenic fate, adding to the astrocyte and oligodendrocyte populations. Using time-lapse video-microscopy of primary cerebral cortex cell cultures, it is possible to study the cellular and molecular mechanisms controlling the mode of cell division and cell cycle parameters of progenitor cells. Similarly, the fate of postmitotic cells can be examined using cell-specific fluorescent reporter proteins or post-imaging immunocytochemistry. More importantly, all these features can be analyzed at the single-cell level, allowing the identification of progenitors committed to the generation of specific cell types. Manipulation of gene expression can also be performed using viral-mediated transfection, allowing the study of cell-autonomous and non-cell-autonomous phenomena. Finally, the use of fusion fluorescent proteins allows the study of symmetric and asymmetric distribution of selected proteins during division and the correlation with daughter cells fate. Here, we describe the time-lapse video-microscopy method to image primary cerebral cortex murine cells for up to several days and analyze the mode of cell division, cell cycle length and fate of newly generated cells. We also describe a simple method to transfect progenitor cells, which can be applied to manipulate genes of interest or simply label cells with reporter proteins.

Live imaging is a powerful tool to study cellular behaviors in real time. Here, we describe a protocol for time-lapse video-microscopy of primary cerebral cortex cells that allows a detailed examination of the phases enacted during the lineage progression from primary neural stem cells to differentiated neurons and glia.

生活成像的脑皮层细胞使用二维培养系统

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or ...

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

Bone marrow-derived macrophages (BMDMs) are mature leukocytes that serve a critical physiological role as professional phagocytes capable of clearing a variety of particles. Normally, BMDMs are restricted from the central nervous system (CNS), but following an injury, they can readily infiltrate. Once within the injured CNS tissue, BMDMs are the primary cell type responsible for the clearance of injury-derived cellular debris, including large quantities of lipid rich myelin debris. The neuropathological ramifications of BMDM infiltration and myelin debris phagocytosis within the CNS are complex and not well understood. The protocols described here, allow for the direct in vitro study of BMDMs in the context of CNS injury. We cover murine BMDM isolation and culture, myelin debris preparation, and assays to assess BMDM myelin debris phagocytosis. These techniques produce robust quantifiable results without the need for significant specialized equipment or materials, yet can be easily customized to meet the needs of researchers.

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

We present methods to assess the phagocytic capacity of primary murine bone marrow-derived macrophages using fluorescently labeled myelin debris and intracellular lipid droplet staining.

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 ...

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc superoxide dismutase 1 (SOD1). The structural instability of SOD1 and the detection of SOD1-positive inclusions in familial-ALS patients supports a potential causal role for misfolded and/or aggregated SOD1 in ALS pathology. In this study, we describe the development of a cell-based assay designed to quantify the dynamics of SOD1 aggregation in living cells by high content screening approaches. Using lentiviral vectors, we generated stable cell lines expressing wild-type and mutant A4V SOD1 tagged with yellow fluorescent protein and found that both proteins were expressed in the cytosol without any sign of aggregation. Interestingly, only SOD1 A4V stably expressed in HEK-293, but not in U2OS or SH-SY5Y cell lines, formed aggregates upon proteasome inhibitor treatment. We show that it is possible to quantify aggregation based on dose-response analysis of various proteasome inhibitors, and to track aggregate-formation kinetics by time-lapse microscopy. Our approach introduces the possibility of quantifying the effect of ALS mutations on the role of SOD1 in aggregate formation as well as screening for small molecules that prevent SOD1 A4V aggregation.

We describe a method to quantify the aggregation of misfolded proteins. Our protocol details lentiviral induced stable cell line generation, automated confocal imaging, and image analysis of protein aggregates. As an illustrative application, we studied the effect of small molecules in promoting SOD1 aggregation in a time- and dose-dependent manner.

活细胞 Sod1 突变蛋白聚集物高含量定量测定方法的建立

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...

一种新的体外活体成像法测定星形胶质细胞的吞噬作用 pH 指标-共轭突

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