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1.
钟鸣
(2023-12-28 19:27):
#paper doi:10.1002/cyto.a.23690 , Best Practices for Preparing a Single Cell Suspension from Solid Tissues for Flow Cytometry
本文是一篇综述，描述了从组织制备单细胞悬液的一般性经验和原则。制备单细胞悬液的本质是消化和降解掉细胞间的连接物，即胶原蛋白、蛋白聚糖和糖蛋白,同时也要注意试剂不能破坏细胞膜的完整性，以保护细胞表面分子的完整性，避免造成表位丢失。
分散酶、胶原酶、透明质酸酶用作将组织解离成小细胞团块，其中分散酶可能会破坏细胞表位。细胞-细胞间存在3种链接：1)闭塞连接、2)通信连接和3)锚定连接，使用胰蛋白酶或木瓜蛋白酶破坏他们。胰蛋白酶会对细胞膜蛋白有非常严重的影响，且会导致游离DNA诱导的细胞聚集，因此要避免使用。一种替代方案是木瓜蛋白酶，但其同样会导致游离 DNA 诱导的细胞聚集。还需要引入DNA酶来降解游离的DNA，通常使用DNase-I而非DNase-II，因为前者不启动细胞凋亡途径。钙离子在这一步是必要的，因其能充当DNA酶的激活剂。
关于酶的使用，确定酶解中所用酶的最佳强度和浓度是经验性的，对于正确分离细胞和成功消化组织至关重要。根据酶的不同，酶解也可以在 4°C或冰上进行，这些较低的温度可能会减慢酶的反应速率并延长潜伏期，但有助于最大限度地减少细胞死亡。
酶解结束后推荐使用流式计数，添加核染色剂可以区分完整细胞和细胞碎片，添加活性染料可以定量死细胞的百分比。如果下游是流式分析且需要保存一段时间，最好使用多聚甲醛固定，特别是对于脆弱/异质群体（例如肺单细胞）。
文章最后还提供了其他有益建议。
Abstract:
Preparing a single cell suspension is a critical step in any solid tissue flow cytometry experiment. Tissue dissection, enzymatic digestion, and mechanical dissociation are three significant steps leading to the …
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Preparing a single cell suspension is a critical step in any solid tissue flow cytometry experiment. Tissue dissection, enzymatic digestion, and mechanical dissociation are three significant steps leading to the degradation of the extracellular matrix and the isolation of single cells, allowing the generation of high-quality flow cytometry data. Cells and the extracellular matrix contain various proteins and other structures which must be considered when designing a tissue digestion protocol to preserve the viability of cells and the presence of relevant antigens while digesting matrix components and cleaving cell-cell junctions. Evaluation of the single cell suspension is essential before proceeding with the labeling of the cells as high viability and absence of cell debris and aggregates are critical for flow cytometry. The information presented should be used as a general guide of steps to consider when preparing a single cell suspension from solid tissues for flow cytometry experiments. © 2018 International Society for Advancement of Cytometry.
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