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

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• 作者：Daniela S. Masson-Meyers ^{masson@uwm.edu" class="auth_mail" title="E-mail the corresponding author} ; Violet V. Bumah ^{bumah@uwm.edu" class="auth_mail" title="E-mail the corresponding author} ; Chukuka S. Enwemeka ; ¹ ; ^{Enwemeka@mail.sdsu.edu" class="auth_mail" title="E-mail the corresponding author}
• 关键词：MTT ; 3-[4 ; 5-dimethylthiazol-2-yl]-2 ; 5-diphenyl tetrazolium bromide ; EthD-1 ; ethidium homodimer-1 ; DMEM ; Dulbecco's modified Eagle's medium ; FBS ; fetal bovine serum ; PBS ; phosphate-buffered saline ; EDTA ; ethylenediaminetetraacetic acid ; LEDs ; light-emitting diodes ; DPBS ; Dulbecco's phosphate-buffered saline ; FITC ; fluorescein isothiocyanate ; TRITC ; tetramethylrhodamine isothiocyanate
• 刊名：Journal of Pharmacological and Toxicological Methods
• 出版年：2016
• 出版时间：May-June 2016
• 年：2016
• 卷：79
• 期：Complete
• 页码：15-22
• 全文大小：1021 K

文摘

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 220 J/cm², incubated for 24 h and viability assessed using each test.

Results

MTT assay showed significant decreases in viability when cells were irradiated with 110 and 220 J/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 220 J/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 220 J/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 3 J/cm² or 55 J/cm² did not adversely affect cell viability. Thus, doses below 110 J/cm² appear safe.