Developmental Biology
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从舌头上皮和梅森奇姆/胚胎日12.5和8周大的老鼠的结缔细胞分离
Chapters
Summary January 21st, 2021
Please note that all translations are automatically generated.
Click here for the English version.
我们开发了一个通用的协议，将大量高质量的单细胞从胚胎和成年小鼠舌头的上皮和间质/结缔组织分离出来。
Transcript
复杂组织细胞的高产量和质量对于常用的实验分析（如单细胞RNA测序和原发干细胞培养）至关重要。该协议产生健康的细胞在高产和质量从不同区域和组织隔间的哺乳动物舌头，包括舌头上皮和结缔组织。要将上皮与E 12.5小鼠舌头的发液分离，将安乐死的怀孕雌性小鼠转移到手术区，用70%乙醇湿润小鼠腹部，以防止毛皮进入手术部位。
用解剖剪刀打开腹部，露出携带胚胎的子宫角，解剖子宫角，并将它们转移到100毫米培养皿中15毫升的新鲜Tyrode溶液中。在解剖显微镜下，使用迷你剪刀和细钳解剖子宫角的胚胎。用细钳打开口腔，用迷你剪刀解剖可施用的舌头。
在 100 毫米培养皿中用 15 毫升新鲜无菌 Tyrode 的溶液清洗舌头。然后将组织转移到35毫米培养皿中两毫升的酶混合物中，在37摄氏度下孵育20分钟。将舌头转移到15毫升的新鲜无菌Tyrode溶液中，然后用细钳轻轻地将心肌和上皮从心室一侧分离。
用15毫升新鲜无菌Tyrode的溶液清洗分离的绰号和梅森奇姆两次。要将舌头上皮与成年小鼠的结缔组织分离，将安乐死小鼠转移到手术区，用70%乙醇弄湿小鼠头部，以防止毛皮进入口腔。使用解剖剪刀沿脸颊切口角，打开口腔。
用可塑性解剖舌头，洗净舌头，并将其放在塑料盘中，并加一层塑料包装。使用手术钳保持舌头，通过后舌头的尖端在舌头的亚皮空间中注入消遣和拼贴酶的酶混合物。将一毫升酶混合物均匀地注射到整个舌头进行组织收集，然后将局部 0 . 5 毫升酶混合物注射到前舌，以便从舌尖或后舌收集组织，用于环状组织收集。
用塑料包装舌头，在37摄氏度下孵育30分钟。使用迷你剪刀解剖包皮纸和舌尖。然后将组织转移到15毫升的新鲜无菌Tyrode溶液中。
根据下游实验的要求，使用迷你剪刀将上皮与酶消化的亚皮空间中的基础结缔组织分离，并将组织修剪成适当的大小。在 100 毫米培养皿中，用 15 毫升新鲜无菌 Tyrode 溶液两次清洗分离的上皮和底层结缔组织。将组织转移到三毫升的0.25%的三肌素EDTA在一个新的35毫米培养皿。
在37摄氏度下将菜孵化30分钟，每5分钟用一毫升移液器尖轻轻搅拌组织。在 DMEM F12 中加入 500 微升 5%FBS，以阻止反应，并将介质转移到 5 毫升低绑定离心机管中。在室温下将细胞悬浮在200倍G下8分钟，并去除超自然。
在含有 10% FBS 和 1% BSA 的 DMEM F12 的三毫升中轻轻恢复细胞，然后用 70 微米细胞过滤器过滤细胞，然后是 35 微米细胞过滤器。在室温下，将细胞悬浮在G的200倍，8分钟，然后取出大部分介质，留下50至300微升作为最后体积来补充细胞。在胚胎小鼠舌头中，在适当的酶消化后，在亚皮空间中可以看到间隙。
在成年小鼠舌头中，通过注射区域的肿胀表示成功的酶注射，这表明酶可以通过舌头保持。在汇集了E12.5舌的表皮板和薄薄的间质层后，手动细胞计数表明，该协议总共产生63，917个细胞，从上皮板存活率为95.2%，从上皮板中产生294，333个细胞，从感性板中存活率为96.3%。当使用 8 周大的 10 个成人舌头时，该协议从舌尖上皮板产生了 187 ， 333 个细胞，其生存能力为 95 . 4% ，从环叶上皮板产生 54 . 4 万个细胞，从环叶酸上皮板产生活性为 96 . 3% 的细胞，从结缔组织中产生 150 ， 500 个活性为 93% 的细胞。
按照此协议，分离的细胞可用于单细胞RNA测序和3D测试器官培养，以定义语言上皮下未识别的测试祖先细胞。
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