Pozarowski Piotr, Huang Xuan, Gong Richard W, Priebe Waldemar, Darzynkiewicz Zbigniew
Brander Cancer Research Institute, New York Medical College, Valhalla, New York, USA.
Cytometry A. 2004 Feb;57(2):113-9. doi: 10.1002/cyto.a.10121.
Common assays of drug-induced cytotoxicity on adherent cells rely on cell trypsinization followed by count of live and dead cells. To estimate the cell cycle effects, cellular DNA content is analyzed by flow cytometry. This procedure is laborious and time consuming. The alternative viability assays, e.g., based on reduction of 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide, although rapid and convenient, do not provide information about individual cells or cell cycle effects and may be biased by growth imbalance.
The bladder carcinoma T-24 cells were seeded onto eight-chamber microscope slide-based tissue culture vessels. The novel antitumor drug, the bis-intercalating anthracycline WP631, was administered at various concentrations to different chamber cultures on the same slide; the control cultures were left untreated. After 24, 48, and 72 h, the cultures were fixed, and cellular DNA was stained with 4,6-diamidino-2-phenyl indole (DAPI). The slides were scanned by laser scanning cytometry (LSC) to obtain the number of attached cells per culture chamber and reveal their cell cycle distribution.
The cell growth and viability plots in the absence and presence of WP621 were constructed from the frequency of the attached cells per chamber. A 50% reduction in cell number was observed at the 75 nM concentration of WP321. Mitotic and postmitotic cells were identified based on high intensity of maximal pixel of DAPI fluorescence. An increase in proportion of cells in G2 was seen at 75-300 nM of WP631. Relatively few (<12%) apoptotic cells, identified by the presence of DNA strand breaks, remained attached in the WP631-treated cultures.
Because late apoptotic cells detach during culturing, the cells that remain attached in the multi-chamber cultures represent predominantly live cells; the deficit in their number compared with the untreated cultures, recorded by LSC during scanning, provides information about the degree of cytostatic and cytotoxic effects of the studied drug. The possibility to demonstrate the cell cycle distribution, including a distinction between G2 and M cells, provides an additional advantage of this assay. Other parameters that may be associated with the cell cycle perturbation or with induction of apoptosis also can be measured in the same cultures by using the multiparameter capabilities of LSC. Each measured cell can be relocated for imaging or measurement after subsequent staining with other probes.
常见的针对贴壁细胞的药物诱导细胞毒性检测方法依赖于细胞胰蛋白酶消化，然后对活细胞和死细胞进行计数。为了评估细胞周期效应，通过流式细胞术分析细胞DNA含量。这个过程既费力又耗时。其他活力检测方法，例如基于3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐还原的方法，虽然快速便捷，但不能提供关于单个细胞或细胞周期效应的信息，并且可能因生长不平衡而产生偏差。
将膀胱癌细胞T-24接种到基于八腔显微镜载玻片的组织培养容器上。将新型抗肿瘤药物双嵌入蒽环类药物WP631以不同浓度施用于同一张载玻片上的不同腔室培养物；对照培养物不进行处理。在24、48和72小时后，固定培养物，并用4,6-二脒基-2-苯基吲哚（DAPI）对细胞DNA进行染色。通过激光扫描细胞术（LSC）扫描载玻片，以获得每个培养腔室中贴壁细胞的数量，并揭示其细胞周期分布。
根据每个腔室中贴壁细胞的频率构建了在有无WP621情况下的细胞生长和活力图。在WP321浓度为75 nM时观察到细胞数量减少了50%。根据DAPI荧光最大像素的高强度鉴定有丝分裂和有丝分裂后细胞。在WP631浓度为75 - 300 nM时，G2期细胞比例增加。通过DNA链断裂的存在鉴定出相对较少（<12%）的凋亡细胞仍附着在WP631处理的培养物中。
由于晚期凋亡细胞在培养过程中会脱落，多腔室培养物中残留的细胞主要代表活细胞；在扫描过程中通过LSC记录的与未处理培养物相比它们数量的减少，提供了有关所研究药物的细胞抑制和细胞毒性作用程度的信息。能够展示细胞周期分布，包括区分G2期和M期细胞，是该检测方法的另一个优势。通过使用LSC的多参数功能，还可以在相同培养物中测量与细胞周期扰动或凋亡诱导相关的其他参数。每个测量的细胞在随后用其他探针染色后可重新定位以进行成像或测量。
