Evaluation of the Alamarblue Assay for Adherent Cell Irradiation Experiments

Abstract

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.

Effect of mitochondrial metabolism-interfering agents on cancer cell mitochondrial function and radio/chemosensitivity

“…The AlamarBlue assay is a reliable method for cell viability [19]. This assay, using the metabolic activity of cells to reduce resazurin (oxidized form: 7-hydroxy-3Hphenoxazin-3-1-10-oxide) to resorufin, counts the number of cells with active mitochondria, given that resazurin reduction is performed by mitochondrial enzymes [20].…”

Assessment of Radiobiological α/β Ratio in Lung Cancer and Fibroblast Cell Lines Using Viability Assays

“…A549, H1299 (250 cells/well) and MRC5 (500 cells/well) cells were seeded in a 96-well plate and irradiated with 0, 2, 4, 6, 9 and 12 Gy. Cell viability was assessed utilizing AlamarBlue ® at 6 and 8 days post irradiation (before surviving cells start their rapid regrowth (6) and before control cells have increased to the upper detectable limit beyond which the AlamarBlue ® method cannot provide cell growth estimates). Thereafter, the dose/viability curves were created and the isoeffect dose D o value (dose that allows 37% viability at 8th day post irradiation) was estimated.…”

“…The AlamarBlue ® assay (ThermoScientific, Waltham, MA USA) is a reliable method for measuring cell viability (6). This assay, using the metabolic activity of cells to reduce resazurin (oxidized form: 7-hydroxy-3H-phenoxazin-3-1-10-oxide) to resorufin, counts the number of cells with active mitochondria, as resazurin reduction is achieved by mitochondrial enzymes (7).…”

“…The relative fluorescence (in RFU units: well minus background units) was measured every 30 min for 12 h, using a microplate reader (FLUOstar ® Omega; BMG LABTECH GmbH, Ortenberg, Germany). Six values for each time point were obtained and the mean was used to plot the dose-response curves (6).…”

Assessment of Nano-toxicity and Safety Profiles of Silver Nanoparticles

“…Fluorescence of resazurin and resorufin can be observed at 530-560 nm stimulation wave length, in addition emission wave length and oxidized form does not fluoresce much at 590 nm. Absorbance value can be observed at 570 and 600 nm, respectively, for the oxidized and reduced forms [124].…”