A comparison of four methods for determining viability in human dermal fibroblasts irradiated with blue light

Introduction: Several tests are available for assessing the viability of cells; however, there is a dearth of studies comparing the results obtained with each test. We compared the capability of four viability assays (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT), neutral red, trypan blue and live/dead fluorescence), to detect potential toxicity in fibroblasts irradiated with 470 nm blue light. Methods: Cells were irradiated at 3, 55, 110 and 220J/cm², incubated for 24h and viability assessed using each test. Results: MTT assay showed significant decreases in viability when cells were irradiated with 110 and 220J/cm² energy fluence (dose) (89% and 57% viable cells, respectively; p<0.0001, compared to control); likewise the trypan blue assay showed 42% and 46% viable cells (p<0.0001). Neutral red assay revealed significant decrease in viability when cells were irradiated with 220J/cm² (84% viable cells; p=0.0008, compared to control). The live/dead fluorescence assay was less sensitive, evincing 91% and 95% viable cells after irradiation with 110 and 220J/cm² respectively. Discussion: (1) The four assays differed in their levels of sensitivity to cell viability. (2) The adverse effect of increasing doses seems to manifest as alteration of mitochondrial metabolism, followed by lysosomal dysfunction, membrane disruption and finally loss of cell membrane integrity. (3) Overall, irradiation with 3J/cm² or 55J/cm² did not adversely affect cell viability. Thus, doses below 110J/cm² appear safe.