Bijman J T, Wagener D J, van Rennes H, Wessels J M, van den Broek P
Cytometry. 1985 Jul;6(4):334-41. doi: 10.1002/cyto.990060410.
The preparation of single-cell suspensions from 25 human head and neck tumors is described. Dispersal was performed overnight at 4 degrees C under slight agitation of the tissue suspensions using various combinations of enzymes and additives. The cell suspensions were examined for number of cells released, viability, amount of debris, and DNA distribution by means of flow cytometry (FCM). It was shown that both trypsin/dithioerythritol (TD) and collagenase/D Nase (CDse) were of value in dispersing single cells from tumor tissue. In contrast to CDse, incubation with TD appeared to be cytolytic to normal lymphocytes. In a number of cases, DNA-FCM revealed ploidy abnormalities in a TD-suspension, which were not discernible in the concurrent CDse-suspension. Cell culture of primary cell suspensions corroborated the reliability of the DNA-FCM measurements. Pretreatment with CDse improved tumor disaggregation by TD and indicated a different dispersal capacity. Addition of Ca2+ and Mg2+ ions to the dispersal mixtures and preincubation of tumor slices in complete medium for 1 day before initiation of cell dispersion influenced favorably the quality of the cell suspension.
本文描述了从25个人类头颈部肿瘤制备单细胞悬液的方法。在4℃下，使用酶和添加剂的各种组合，对组织悬液进行轻微搅拌，分散过夜。通过流式细胞术（FCM）检测细胞悬液中释放的细胞数量、活力、碎片量和DNA分布。结果表明，胰蛋白酶/二硫赤藓糖醇（TD）和胶原酶/D Nase（CDse）在从肿瘤组织中分散单个细胞方面均有价值。与CDse不同，用TD孵育似乎对正常淋巴细胞具有细胞溶解作用。在一些情况下，DNA-FCM显示TD悬液中存在倍体异常，而在同时的CDse悬液中则无法识别。原代细胞悬液的细胞培养证实了DNA-FCM测量的可靠性。用CDse预处理可改善TD对肿瘤的解离作用，并显示出不同的分散能力。在分散混合物中添加Ca2+和Mg2+离子，以及在开始细胞分散前将肿瘤切片在完全培养基中预孵育1天，对细胞悬液的质量有有利影响。
