Pallavicini M G, Folstad L J, Dunbar C
Cytometry. 1981 Sep;2(2):54-8. doi: 10.1002/cyto.990020203.
We used a bacterial neutral protease to disperse KHT solid tumors into single cell suspensions suitable for routine cell kinetic analysis by flow cytometry and for clonogenic cell survival. Neutral protease disaggregation under conditions which would be suitable for routine tumor dispersal was compared with a trypsin/DNase procedure. Cell yield, clonogenic cell survival, DNA distributions of untreated and drug-perturbed tumors, rates of radioactive precursor incorporation during the cell cycle, and preferential cell cycle phase-specific cell loss were investigated. Tumors dispersed with neutral protease yielded approximately four times more cells than those dispersed with trypsin/DNase and approximately a 1.5-fold higher plating efficiency in a semisolid agar system. Quantitative analysis of DNA distributions obtained from untreated and cytosine-arabinoside-perturbed tumors produced similar results with both dispersal procedures. The rates of incorporation of tritiated thymidine during the cell cycle were also similar with neutral protease and trypsin/DNase dispersal. Preferential phase-specific cell loss was not observed with either technique. We find that neutral protease provides good single cell suspensions of the KHT tumor for cell survival measurements and for cell kinetic analysis of drug-induced perturbations by flow cytometry. In addition, the high cell yields facilitate electronic cell sorting where large numbers of cells are often required.
我们使用一种细菌中性蛋白酶将KHT实体瘤分散成单细胞悬液，这种悬液适用于通过流式细胞术进行常规细胞动力学分析以及克隆形成细胞存活分析。将在适合常规肿瘤分散的条件下进行的中性蛋白酶分解与胰蛋白酶/脱氧核糖核酸酶方法进行了比较。研究了细胞产量、克隆形成细胞存活率、未处理和药物干扰肿瘤的DNA分布、细胞周期中放射性前体掺入率以及细胞周期阶段特异性细胞丢失率。用中性蛋白酶分散的肿瘤产生的细胞比用胰蛋白酶/脱氧核糖核酸酶分散的肿瘤多约四倍，并且在半固体琼脂系统中的平板接种效率高约1.5倍。对从未处理和经阿糖胞苷干扰的肿瘤中获得的DNA分布进行定量分析，两种分散方法得到的结果相似。用中性蛋白酶和胰蛋白酶/脱氧核糖核酸酶分散时，细胞周期中氚标记胸腺嘧啶核苷的掺入率也相似。两种技术均未观察到阶段特异性细胞丢失。我们发现，中性蛋白酶可为KHT肿瘤提供良好的单细胞悬液，用于细胞存活测量以及通过流式细胞术对药物诱导的干扰进行细胞动力学分析。此外，高细胞产量便于进行电子细胞分选，因为通常需要大量细胞。
