Assay of anticancer drugs in tissue culture: Comparison of a tetrazolium-based assay and a protein binding dye assay in short-term cultures derived from human malignant glioma
Assay of anticancer drugs in tissue culture: Comparison of a tetrazolium-based assay and a protein binding dye assay in short-term cultures derived from human malignant glioma
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DOI：
10.1097/00001813-199605000-00014
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发表时间：
1996-05-01
影响因子：
2.3
通讯作者：
Darling, JL
中科院分区：
文献类型：
--
作者：
Haselsberger, K;Peterson, DC;Darling, JL
由于MTT法在筛选人恶性胶质瘤短期培养物中存在方法学困难，因此基于磺酰罗丹明B（SR B）蛋白质染色的化疗敏感性测定法已被优化用于这些细胞。对于大多数细胞系，在固定染料浓度下的SRB在20分钟时达到最大染色密度，并且通过使用高于0.2%的染料浓度，该强度不会进一步增加。固定后延迟染色不会显著降低染色强度，但固定和染色后延迟染料提取会降低染色强度。SRB试验或细胞计数测定的细胞数之间存在良好的定量和定性线性关系，但MTT试验始终低估了试验板中的细胞数。MTT试验似乎不能检测少于约150个细胞/孔，而这些少量细胞可通过细胞计数或SRB染色容易地检测到。当终点取为ID 25、ID 50或ID 75时，由MTT和SRB试验获得的丙卡巴肼、CCNU和长春新碱的化学敏感性值之间存在密切相关性。结果表明，SRB能够产生与MTT测定大致相似的结果，但在检测少量细胞时更灵敏，在宽范围的细胞数内细胞数与SRB染色强度之间呈线性关系，它能够从短时间内产生数据-从恶性神经胶质瘤的长期培养物，并提供了技术上的优势，在MTT测定法，板可以安全地储存在某些点在测定过程中，而不需要立即处理。SRB试验提供了一个有用的替代MTT试验，用于确定短期培养的人脑胶质瘤细胞毒药物的敏感性。
Because of the methodological difficulties associated with the MTT assay in screening short-term cultures derived from human malignant glioma, a chemosensitivity assay based on the protein staining using sulforhodamine B (SRB) has been optimized for use with these cells. SRB at a fixed dye concentration achieved maximal staining density at 20 min for most cell lines and this intensity was not further increased by using dye concentrations above 0.2%. A delay in staining after fixation did not significantly decrease staining intensity, but delay in dye extraction after fixation and staining did, There was an excellent quantitative and qualitative linear relationship between cell number determined by either the SRB essay or by cell counting, but not with the MTT assay which consistently underestimated the number of cells in assay plates. The MTT assay appeared to be incapable of detecting less than about 150 cells/well, while these small numbers of cell were readily detectable by either cell counting or SRB staining, There was a close correlation between chemosensitivity values derived from the MTT and SRB assays for procarbazine, CCNU and vincristine when the endpoint is taken as either the ID25, ID50 or ID75. The results indicate that the SRB is capable of producing broadly similar results to the MTT assay, but is more sensitive in the detection of small numbers of cells with a linear relationship between cell number and SRB staining intensity over a wide range of cell numbers, It is capable of producing data from short-term cultures from malignant glioma and offers technical advantages over the MTT assay in that plates may safely be stored at certain points during the assay without the need for immediate processing. The SRB assay provides a useful alternative to the MTT assay for determining the sensitivity of short-term cultures of human glioma to cytotoxic drugs.