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小鼠脑海马组织机械和酶解离联合
Chapters
Summary October 21st, 2021
Please note that all translations are automatically generated.
Click here for the English version.
该神经细胞解离方案适用于起始材料量低的样品，并产生用于下游分析的高活性单细胞悬浮液，并具有可选的固定和染色步骤。
Transcript
成功解离少量神经组织可以使实验室获得对治疗效果，细胞功能以及疾病和治疗作用机制的结构特异性见解。这种神经解离方案始终如一地产生高度可行和实际的单细胞悬浮液。此外，我们可以处理的样品只是商业试剂盒所针对样品的一小部分。
适当的规划和准备是该技术成功的关键。运行几轮练习以熟悉协议将是有益的。麻醉一只六个月大的雌性C57BL / 6J小鼠后，捏住下腹部并使用镊子抬起皮肤。
然后用剪刀，穿过皮毛和皮肤到肋骨底部。做两个对角线切口，从胸腔下方开始，向每个肩膀移动。然后小心地切除横膈膜和胸腔以暴露心脏。
小心地去除心脏周围的任何结缔组织后，用剪刀夹住右心房。然后关闭异氟醚流向呼吸器。用镊子和蝴蝶针的斜面朝上保持心脏稳定，刺穿左心室，同时保持针头水平并与动物平行。
接下来，将针头固定到位，打开泵并灌注至少30毫升盐水或肝素溶液，直到离开心脏的液体不透明，肝脏和肺部颜色苍白。灌注后，关闭泵，取出针头，然后将小鼠转移到解剖区域。斩首后，将皮毛从后脑勺切开到眼睛，然后将皮肤剥开以露出头骨。
接下来，将头骨夹在眼睛之间，并在10点钟和2点钟位置的头骨背面做两个切口。然后沿着颅骨的中鼓弦线进行一个长切口，直到眼睛之间的原始切口。接下来，使用镊子将头骨的两半剥开到两侧。
然后使用刮刀去除大脑并将其放入装有冷DPBS的60毫米玻璃培养皿中并保持在冰上。使用手术刀或剃须刀，分离每个半球并去除嗅球和小脑。然后切除中脑，直到海马体暴露出来。
接下来，用镊子保护大脑。然后使用第二组镊子，轻轻地将海马体从每个半球中梳理出来。将两个海马体转移到含有冷DPBS的标记的1.5毫升管中，并将管放在冰上。
使用镊子将海马组织碎片转移到C管中，并向管中加入30微升酶混合物2。拧紧盖子并拧紧直至发出咔嗒声后，将管子放入解离器中并运行相应的程序。在程序运行时，用两毫升BSA缓冲液预润一个70微米的细胞过滤器，放置在50毫升的锥形管上。
解离程序完成后，向解离组织中加入四毫升BSA缓冲液，并通过50毫升锥形管上的细胞过滤器过滤混合物。接下来，向C管中加入10毫升DPBS。然后关闭管，轻轻旋转溶液，并通过50毫升锥形管上的细胞过滤器过滤。
离心过滤的细胞悬浮液，丢弃上清液而不干扰沉淀并储存沉淀。为了去除碎屑，用1， 550微升冷DPBS重新悬浮沉淀，并将悬浮液转移到标记的50毫升锥形管中。然后加入450微升冷碎屑去除溶液，上下移液。
用一毫升冷DPBS轻轻覆盖细胞悬浮液，使尖端保持在锥形管壁上。重复该过程，直到总叠加量为两毫升。接下来，将悬浮液以3， 000倍G离心10分钟，完全加速和完全断裂。
吸出最顶层，然后来回扫动移液器尖端以吸出白色中间层。尽可能多地去除中间层，而不干扰最底层。接下来，加入两毫升冷的DPBS，上下移液以混合。
然后将悬浮液以1， 000倍G离心10分钟，完全加速和完全断裂。离心后，弃去上清液并将沉淀重悬于一毫升BSA缓冲液中。细胞计数后，离心剩余的细胞悬浮液并将沉淀重悬于50微升稀释的活死渍中。
然后将样品转移到标记的流管中，并在室温下在黑暗中孵育8至10分钟。孵育后，加入500微升BSA缓冲液并重复离心步骤。然后丢弃上清液，在管中留下少量缓冲液。
主栅极排除了前向散射图与侧散点图中的碎片，随后排除了死细胞。第二个门排除了髓鞘碱性蛋白阳性的细胞。在剩余的细胞中，创建了每个荧光染料阳性细胞的密度图。
每个神经元细胞群的频率是从第三门计算出来的。使用手动机械解离和酶消化处理的样品产生的目标细胞群大幅减少，而使用自动机械解离和酶消化处理的新鲜和固定样品都显示出目标细胞群的几倍高。虽然灌注和碎屑清除步骤最初可能具有挑战性，但保持手部光滑稳定有助于确保成功。
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