Name: a novel in vitro live-imaging assay of astrocyte-mediated phagocytosis using ph indicator-conjugated synaptosomes
Uploaded: 2018-02-05T13:30:00.000+00:00
Duration: 6 min 43 s
Description: 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

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。
为了成功地进行这一实验, 需要在洗涤步骤中仔细交换介质和 DPBS (5 节)。原代培养的星形胶质细胞和小胶质, 特别是胶质, 容易暴露于空气中。因此, 减少洗涤步骤之间的时间间隔对于维持活细胞是至关重要的。对于使用活成像仪器建立长期实时成像, 建议手动对焦, 因为自动对焦可能会增加激光/LED 曝光时间, 这可能会损坏细胞。
在这种方法中, 体纯化协议19略有修改。原纸将渐变解决方案分为3%、10%、15% 和23%。然而, 我们建立了3%、10% 和23% 的梯度溶液来提高纯化突的收率。该方法还对 immunopanning 星形胶质细胞和胶质电池的说明进行了修改。原始平移协议21的目标是仅纯化星形胶质细胞, 并使用 BSL-1、次只、CD45、O4 和 Itgb5 (HepaCAM) 作为 immunopanning 顺序。由于 BSL-1 和二次唯一的板将去除内皮细胞和小胶质, 我们改变了顺序对 CD45, 仅二次, BSL-1, O4 和 Itgb5 (HepaCAM 为老鼠) 增加从 CD45 平移盘的微胶质的产量。在本协议中, 我们还通过循环系统将 SD 大鼠幼崽 (~ P7-P10) 灌入 DPBS, 以去除各种血细胞, 以减少 CD45-positive 非胶质人群的污染。
有几个优点的体外吞噬试验。用于体共轭的 ph 值指标只在低 ph 条件下发出红光荧光。因此, 在处理数据时, 我们可以直接将红色荧光作为吞噬事件的信号, 而无需淬火程序来去除背景信号或复杂的成像分析, 以获得被吞噬物质的 co 局部化信号。吞噬.此外, 这种方法允许实时跟踪的胶质吞噬的图像 pH 指示强度在一个给定的 ROI 在多个时间点。通过生成 pH 值指示强度高达 100 h 的图, 可以很容易地在不同的细胞和条件之间监测吞没以及降解能力。另一个优点是使用突。由于星形胶质细胞 ensheathe 突触的所有时间与他们的精细过程, 并已被证明吞噬突触在体内4, 使用突是非常适合的测量体外吞噬能力的星形胶质细胞。最后, 这个体外吞噬试验有可能发展到研究胶质细胞吞噬髓鞘碎片或淀粉样β, 这是与各种神经紊乱有关。
随着预期寿命的延长, 神经退行性疾病患者的数量急剧增加是不可避免的。神经胶质细胞的突触丢失是几种神经退行性疾病的主要因素之一13,14。此外, 异常的突触修剪和脑内稳态失衡可能与神经胶质细胞吞噬缺陷有关。因此, 识别因素和化合物, 可以控制吞没和降解能力的星形胶质细胞可能是关键的寻找成功的治疗各种神经疾病。由于这种体外吞噬试验可以很容易地扩大与井板, 这种方法可以作为一个合适的平台, 各种放映, 以确定这些因素。

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>&lt;strong&gt;Synaptosome purification&lt;/strong&gt;</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>&lt;strong&gt;pH indicator conjugation&lt;/strong&gt;</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>&lt;strong&gt;Immunopanning&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>10X Earle&amp;rsquo;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 (&amp;mu;-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&amp;amp;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.&lt;sup class=&quot;xref&quot;&gt;23&lt;/sup&gt;</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 &amp;mu;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>&lt;strong&gt;Collect IP-ACM&lt;/strong&gt;</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>&lt;strong&gt;Phagocytosis live imaging assay&lt;/strong&gt;</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胞吞噬时发出明亮的红色荧光。实时比较胶质细胞的吞没和降解能力, 可以采取每1或2小时 (补充电影 1,补充电影 2) 的多个 ROI 图像。用这种方法, 我们展示了星形胶质细胞和小胶质细胞介导的吞噬的不同动力学 (图 6)。为了量化胶质细胞的吞噬功能, 测量了红色荧光信号的面积 (µm2), 在图 6B和 图 7中被称为吞噬指数。虽然星形胶质细胞在吞噬大量的 pH 指标-共轭突中似乎是有效的, 但在吞噬和降解突 (图 6B) 时, 小胶质组织的速度更快。小胶质细胞显示最高 ph 值指示强度在26小时后, ph 指标-共轭体治疗, 而星形胶质显示他们的最大值为45小时 (p-价值 < 0.05, 双向方差分析与 1X acm 和小胶质组织与 1X acm)。同样, 胶质细胞在峰值点后40小时的总 ph 值指示强度降低了 20.7%, 而星形胶质细胞膜在同一时间段的总 ph 值显示降低了17%。有趣的是, 我们的数据显示, 含有在 ACM 中的星形胶质细胞的分泌因子, 对于增加星形胶质星和小胶质细胞介导的吞噬作用至关重要 (图 6B)。星形胶质细胞已被证明可以释放桥接分子, 如 MFGE8、GAS6 和蛋白质, 它们能够弥合和诱导吞噬受体与 "吃我" 信号 (如 PS23) 之间的相互作用。如上所述, 星形胶质细胞通过 MERTK 和 MEGF10 通路4消除突触。MEGF10 只由大脑中的星形胶质细胞表达, 通过识别具有未知特征的 "吃-我" 信号参与突触吞没。与以往的研究结果一致, 该检测结果表明, 与野生型 (小鼠) 星形胶质细胞相比, Megf10敲出 (KO) 小鼠星形胶质细胞具有明显的吞噬能力 (图 7)。与重量型星形胶质细胞相比, Megf10高星形胶质细胞在峰值点 (31 h) (图 7) 中的 pH 值指示强度降低了约40%。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56647/56647fig1.jpg"> 图1。用 immunopanning 方法纯化星形胶质细胞的示意图.<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56647/56647fig1large.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/56647/56647fig2.jpg"> 图2。吞噬活成像试验原理图.<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56647/56647fig2large.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/56647/56647fig3.jpg"> 图3。在梯度溶液中具有代表性的脑匀浆分馏.<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56647/56647fig3large.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/56647/56647fig4.jpg"> 图4。PS 暴露的突的代表性图像.(a) 突星形胶质细胞的明亮场图像。突附着在星形胶质细胞上。(B) tdTomato 阳性突的荧光图像, 从 tdTomato 表达的鼠脑中纯化。(C) pSIVA 绑定到 PS, 这是暴露在体外膜, 并发出绿色荧光。(D) 由 pSIVA (绿色) 检测到的 PS 与 tdTomato-正突 (红色) 进行了联合本地化。缩放栏:20 µm<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56647/56647fig4large.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/56647/56647fig5.jpg"> 图 5。具有代表性的星形胶质细胞的明亮场和荧光图像--共轭突在两个时间点。在治疗后11小时 (底部板), pH 指标-共轭突被吞噬的星形胶质细胞和发出红色荧光, 而他们不在1小时后治疗 (上部面板)。缩放栏:50 µm.<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56647/56647fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56647/56647fig6.jpg"> 图 6。大鼠星形胶质细胞的吞噬动力学及长期活成像。(a) 使用纯化的星形胶质细胞和小胶质的体外吞噬试验的原理图, 以及 pH 指示物共轭突。请注意, 在拍摄活动图像之前, 未绑定的突在40分钟的孵化后被冲走。(B) 具有代表性的图, 显示星形胶质细胞和小胶质吞没和降解动力学。含有星形胶质细胞分泌因子的 ACM 能显著增强星形胶质细胞和小胶质细胞介导的体吸收。星形胶质细胞: 控制与含 ACM 1X 的星形胶质细胞, ***, Tukey 的多重比较试验。小胶质细胞: 控制与含 ACM 1X 的小胶质细胞, ***, Tukey 的多重比较试验。误差线指示 S.E.M. *p ≤ 0.05, *** p ≤ 0.0001, 双向方差分析。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56647/56647fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56647/56647fig7.jpg"> 图7。减少吞噬能力的Megf10高鼠星形胶质细胞与 1X acm 相比, 小鼠星形胶质细胞与 1X acm.重量与Megf10高星形胶质细胞与 1X ACM, ***, 双向方差分析。误差线指示 S.E.M. * p ≤ 0.05, ** p ≤ 0.01, ** p ≤0.001 双向方差分析。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56647/56647fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
表 1: 解决方案配方.<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56647/JoVE_Materials_R.xls">请单击此处下载此文件.</a>
补充电影1。一种具有代表性的活成像视频, 显示突星形胶质细胞对 pH 指示物的吞噬功能。<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56647/Supplementary_data_Movie_1A_Astrocytes.mp4">请单击此处下载此文件.</a>
补充电影2。一种具有代表性的活成像视频, 显示了 pH 指示物的吞噬作用-胶质细胞共轭突.<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56647/Supplementary_data_Movie_1B_Microglia.mp4">请单击此处下载此文件.</a>

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

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

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

12.1K 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

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 ...

科研

神经科学

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

三维组织工程化排列的星形胶质细胞网络, 以概括发展机制和促进神经系统再生

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

生物工程

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

评估视网膜小胶质细胞吞噬功能<em&gt;在体内</em&gt;使用基于流式细胞术流量测定

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

免疫与感染

Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions

Ischemic stroke is a complex brain injury caused by a thrombus or embolus obstructing blood flow to parts of the brain. This leads to deprivation of oxygen and glucose, which causes energy failure and neuronal death. After an ischemic stroke insult, astrocytes become reactive and proliferate around the injury site as it develops. Under this scenario, it is difficult to study the specific contribution of astrocytes to the brain region exposed to ischemia. Therefore, this article introduces a methodology to study primary astrocyte reactivity and proliferation under an in vitro model of an ischemia-like environment, called oxygen glucose deprivation (OGD). Astrocytes were isolated from 1-4 day-old neonatal rats and the number of non-specific astrocytic cells was assessed using astrocyte selective marker Glial Fibrillary Acidic Protein (GFAP) and nuclear staining. The period in which astrocytes are subjected to the OGD condition can be customized, as well as the percentage of oxygen they are exposed to. This flexibility allows scientists to characterize the duration of the ischemic-like condition in different groups of cells in vitro. This article discusses the timeframes of OGD that induce astrocyte reactivity, hypertrophic morphology, and proliferation as measured by immunofluorescence using Proliferating Cell Nuclear Antigen (PCNA). Besides proliferation, astrocytes undergo energy and oxidative stress, and respond to OGD by releasing soluble factors into the cell medium. This medium can be collected and used to analyze the effects of molecules released by astrocytes in primary neuronal cultures without cell-to-cell interaction. In summary, this primary cell culture model can be efficiently used to understand the role of isolated astrocytes upon injury.

Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions

Ischemic stroke is a complex event in which the specific contribution of astrocytes to the affected brain region exposed to oxygen glucose deprivation (OGD) is difficult to study. This article introduces a methodology to obtain isolated astrocytes and study their reactivity and proliferation under OGD conditions.

在缺血样条件下监测星形胶质细胞的反应性和增殖体外

Ischemic stroke is a complex brain injury caused by a thrombus or embolus obstructing blood flow to parts of the brain. This leads to deprivation of ...

Visualizing and Analyzing Intracellular Transport of Organelles and Other Cargos in Astrocytes

Astrocytes are among the most abundant cell types in the adult brain, where they play key roles in a multiplicity of functions. As a central player in brain homeostasis, astrocytes supply neurons with vital metabolites and buffer extracellular water, ions, and glutamate. An integral component of the &#8220;tri-partite&#8221; synapse, astrocytes are also critical in the formation, pruning, maintenance, and modulation of synapses. To enable these highly interactive functions, astrocytes communicate among themselves and with other glial cells, neurons, the brain vasculature, and the extracellular environment through a multitude of specialized membrane proteins that include cell adhesion molecules, aquaporins, ion channels, neurotransmitter transporters, and gap junction molecules. To support this dynamic flux, astrocytes, like neurons, rely on tightly coordinated and efficient intracellular transport. Unlike neurons,&#160;where intracellular trafficking has been extensively delineated, microtubule-based transport in astrocytes has been less studied. Nonetheless, exo- and endocytic trafficking of cell membrane proteins and intracellular organelle transport orchestrates astrocytes&#8217; normal biology, and these processes are often affected in disease or in response to injury. Here we present a straightforward protocol to culture high quality murine astrocytes, to fluorescently label astrocytic proteins and organelles of interest, and to record their intracellular transport dynamics using time-lapse confocal microscopy. We also demonstrate how to extract and quantify relevant transport parameters from the acquired movies using available image analysis software (i.e., ImageJ/FIJI) plugins.

Here we describe an in vitro live-imaging method to visualize intracellular transport of organelles and trafficking of plasma membrane proteins in murine astrocytes. This protocol also presents an image analysis methodology to determine cargo transport itineraries and kinetics.

可视化和分析星形细胞中细胞内器官和其他货物的细胞内传输

Astrocytes are among the most abundant cell types in the adult brain, where they play key roles in a multiplicity of functions. As a central player in ...

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. ...

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple neurological diseases. Studying human microglia in health and disease represents a challenge due to the extremely limited supply of human cells. Induced pluripotent stem cells (iPSCs) derived from human individuals can be used to circumvent this barrier. Here, it is demonstrated how to differentiate human iPSCs into microglia-like cells (iMGs) for in vitro experimentation. These iMGs exhibit the expected and physiological properties of microglia, including microglia-like morphology, expression of proper markers, and active phagocytosis. Additionally, documentation for isolating and labeling synaptosome substrates derived from human iPSC-derived lower motor neurons (i3LMNs) is provided. A live-cell, longitudinal imaging assay is used to monitor engulfment of human synaptosomes labeled with a pH-sensitive dye, allowing for investigations of iMG's phagocytic capacity. The protocols described herein are broadly applicable to different fields that are investigating human microglia biology and the contribution of microglia to disease.

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

This protocol describes the differentiation process of human induced pluripotent stem cells (iPSCs) into microglia-like cells for in vitro experimentation. We also include a detailed procedure for generating human synaptosomes from iPSC-derived lower motor neurons that can be used as a substrate for in vitro phagocytosis assays using live-cell imaging systems.

人小胶质细胞样细胞:使用人突触体从诱导多能干细胞和 体外 活细胞吞噬测定中分化

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple ...

基于流式细胞术的突触材料小胶质细胞吞噬检测

Quantification of Microglial Engulfment of Synaptic Material Using Flow Cytometry

Microglia play a pivotal role in synaptic refinement in the brain. Analysis of microglial engulfment of synapses is essential for comprehending this process; however, currently available methods for identifying microglial engulfment of synapses, such as immunohistochemistry (IHC) and imaging, are laborious and time-intensive. To address this challenge, herein we present in vitro and in vivo* assays that allow fast and high-throughput quantification of microglial engulfment of synapses using flow cytometry.
In the in vivo* approach, we performed intracellular vGLUT1 staining following fresh cell isolation from adult mouse brains to quantify engulfment of vGLUT1+ synapses by microglia. In the in vitro synaptosome engulfment assay, we used freshly isolated cells from the adult mouse brain to quantify the engulfment of pHrodo&#160;Red-labeled synaptosomes by microglia. These protocols together provide a time-efficient approach to quantifying microglial engulfment of synapses and represent promising alternatives to labor-intensive image analysis-based methods. By streamlining the analysis, these assays can contribute to a better understanding of the role of microglia in synaptic refinement in different disease models.

作者聚焦：通过心理神经免疫学的技术创新促进理解

Here we present two protocols to quantify microglial engulfment of vGLUT1-positive synapses and pHRodo&#160;Red-labeled crude synaptosomes using flow cytometry.

使用流式细胞术定量突触材料的小胶质细胞吞噬

Microglia play a pivotal role in synaptic refinement in the brain. Analysis of microglial engulfment of synapses is essential for comprehending this ...

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

摘要

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此视频中的章节

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

三维组织工程化排列的星形胶质细胞网络, 以概括发展机制和促进神经系统再生

评估视网膜小胶质细胞吞噬功能

在缺血样条件下监测星形胶质细胞的反应性和增殖体外

可视化和分析星形细胞中细胞内器官和其他货物的细胞内传输

iPSC-巨噬细胞死亡神经母细胞的体外定量成像检测

人小胶质细胞样细胞:使用人突触体从诱导多能干细胞和体外活细胞吞噬测定中分化

使用流式细胞术定量突触材料的小胶质细胞吞噬

使用流式细胞术定量突触材料的小胶质细胞吞噬

使用流式细胞术定量突触材料的小胶质细胞吞噬

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

摘要

探索更多视频

此视频中的章节

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

三维组织工程化排列的星形胶质细胞网络, 以概括发展机制和促进神经系统再生

评估视网膜小胶质细胞吞噬功能

在缺血样条件下监测星形胶质细胞的反应性和增殖体外

可视化和分析星形细胞中细胞内器官和其他货物的细胞内传输

iPSC-巨噬细胞死亡神经母细胞的体外定量成像检测

人小胶质细胞样细胞:使用人突触体从诱导多能干细胞和 体外 活细胞吞噬测定中分化

使用流式细胞术定量突触材料的小胶质细胞吞噬

使用流式细胞术定量突触材料的小胶质细胞吞噬

使用流式细胞术定量突触材料的小胶质细胞吞噬

人小胶质细胞样细胞:使用人突触体从诱导多能干细胞和体外活细胞吞噬测定中分化