Department of Radiotherapy / Oncology, Radiobiology and Radiopathology Unit, Democritus University of Thrace, Alexandroupolis, Greece.
Dose Response. 2013 Sep 12;12(2):246-58. doi: 10.2203/dose-response.13-024.Koukourakis. eCollection 2014 May.
The AlamarBlue assay is based on fluorometric detection of metabolic mitochondrial activity of cells. In this study, we determined the methodology for application of the assay to radiation response experiments in 96-well plates. AlamarBlue was added and its reduction measured 7 hours later. Selection of the initial number of plated cells was important so that the number of proliferating cells remains lower than the critical number that produced full AlamarBlue reduction (plateau phase) at the time points of measurements. Culture medium was replaced twice a week to avoid suppression of viability due to nutrient competition and metabolic waste accumulation. There was no need to replace culture medium before adding AlamarBlue. Cell proliferation continued after irradiation and the suppression effect on cell viability was most evident on day 8. At this time point, by comparing measurements from irradiated vs. non-irradiated cells, for various dose levels, a viability dose response curve was plotted. Immediately after the 8(th) day (nadir), cells started to re-grow at a rate inversely related to the radiation dose. By comparing measurements at the time point of nadir vs. a convenient subsequent time point, re-growth dose response abilities were plotted, simulating clonogenic assays.
AlamarBlue 检测法基于细胞代谢线粒体活性的荧光检测。在这项研究中，我们确定了将该检测法应用于 96 孔板中辐射反应实验的方法。加入 AlamarBlue 并在 7 小时后测量其还原量。选择初始接种细胞的数量很重要，以便在测量时间点，增殖细胞的数量仍低于产生完全 AlamarBlue 还原（平台期）的临界数量。每周更换两次培养基，以避免由于营养竞争和代谢废物积累而抑制细胞活力。添加 AlamarBlue 之前不需要更换培养基。细胞增殖在照射后继续进行，并且对细胞活力的抑制作用在第 8 天最为明显。此时，通过比较照射和未照射细胞的测量值，针对不同的剂量水平，绘制细胞活力剂量反应曲线。在第 8 天（最低点）之后，细胞立即开始以与辐射剂量成反比的速度重新生长。通过比较最低点与方便的后续时间点的测量值，绘制再生长剂量反应能力，模拟集落形成测定法。
