Cellular Pathway Imaging Laboratory (CPIL), Molecular Imaging Program at Stanford, Stanford University School of Medicine, 3155 Porter Drive, Palo Alto, CA, 94305, USA.
Laboratory of Experimental and Molecular Neuroimaging (LEMNI), Molecular Imaging Program at Stanford, Stanford University School of Medicine, 300 Pasteur Drive, Grant S-031, Stanford, CA, 94305, USA.
Sci Rep. 2018 Nov 5;8(1):16363. doi: 10.1038/s41598-018-34696-x.
Cell-based assays that measure anticancer drug effects are essential for evaluating chemotherapeutic agents. Many assays targeting various cellular mechanisms are available, leading to inconsistent results when using different techniques. We critically compared six common assays, as well as a new assay using Bodipy.FL.L-cystine (BFC), to identify the most accurate and reproducible in measuring anticancer drug effects. We tested three common chemotherapies (methotrexate, paclitaxel, and etoposide) in two cell lines (Ln229 and MDA-MB231). Spectroscopic assays such as Cell Titer Blue, and 2',7'-dichlorofluorescin diacetate (DCFDA) yielded a strong drug dose response, especially for paclitaxel and etoposide (R = 0.9). MTT and Calcein-AM fluorescent dye-based assays were less consistent in that regard. Among three flow cytometry assays, Propidium Iodide (PI)-based DNA content analysis and a new BFC-based glutathione-redox (GSH) assay produced drug dose dependent results. Compared to PI, BFC showed a better correlation (R = 0.7-0.9) in depicting live and apoptotic cells. We found that the combination of Cell Titer Blue spectroscopy and BFC flow cytometry assays were most accurate in assessing anticancer drug effects by clear distinction between live and apoptotic cells, independent of drug mechanism of action. We present a new application of BFC as an agent for measuring cellular apoptosis.
基于细胞的抗癌药物效果测定法对于评估化学治疗剂至关重要。有许多针对各种细胞机制的测定法，而使用不同技术会导致结果不一致。我们对六种常见的测定法进行了严格比较，还使用了一种新的基于 Bodipy.FL.L-胱氨酸 (BFC) 的测定法，以确定在测量抗癌药物效果方面最准确和最可重复的方法。我们在两种细胞系（Ln229 和 MDA-MB231）中测试了三种常见的化疗药物（甲氨蝶呤、紫杉醇和依托泊苷）。光谱测定法，如 Cell Titer Blue 和 2'，7'-二氯荧光素二乙酸酯（DCFDA），产生了强烈的药物剂量反应，特别是对于紫杉醇和依托泊苷（R = 0.9）。MTT 和 Calcein-AM 荧光染料测定法在这方面不太一致。在三种流式细胞术测定法中，碘化丙啶（PI）基于 DNA 含量分析和新的基于 BFC 的谷胱甘肽-氧化还原（GSH）测定法产生了依赖于药物剂量的结果。与 PI 相比，BFC 在描绘活细胞和凋亡细胞方面显示出更好的相关性（R = 0.7-0.9）。我们发现，通过清楚地区分活细胞和凋亡细胞，Cell Titer Blue 光谱和 BFC 流式细胞术测定法的组合在评估抗癌药物效果方面最准确，而与药物作用机制无关。我们提出了 BFC 作为测量细胞凋亡的新应用。
