title: A comparison of four methods for determining viability in human dermal fibroblasts irradiated with blue light.
abstract: BACKGROUND
  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 470nm blue light.
  
  
  METHODS
  Cells were irradiated at 3, 55, 110 and 220J/cm(2), 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/cm(2) 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/cm(2) (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/cm(2) respectively.
  
  
  CONCLUSIONS
  (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/cm(2) or 55J/cm(2) did not adversely affect cell viability. Thus, doses below 110J/cm(2) appear safe.
authors.affiliation: College of Health Sciences, University of Wisconsin-Milwaukee, 2400 East Hartford Ave., Milwaukee, WI 53211, USA. Electronic address: masson@uwm.edu.
authors.forename: Daniela S
authors.initials: DS
authors.lastname: Masson-Meyers
authors.affiliation: College of Health Sciences, University of Wisconsin-Milwaukee, 2400 East Hartford Ave., Milwaukee, WI 53211, USA. Electronic address: bumah@uwm.edu.
authors.forename: Violet V
authors.initials: VV
authors.lastname: Bumah
authors.affiliation: College of Health Sciences, University of Wisconsin-Milwaukee, 2400 East Hartford Ave., Milwaukee, WI 53211, USA. Electronic address: Enwemeka@mail.sdsu.edu.
authors.forename: Chukuka S
authors.initials: CS
authors.lastname: Enwemeka
mesh_terms: D002470:Cell Survival / Q000502:physiology*; D002478:Cells, Cultured; D005347:Fibroblasts / Q000502:physiology*; D006801:Humans; D008027:Light / Q000009:adverse effects*; D008928:Mitochondria / Q000378:metabolism / Q000502:physiology; D012867:Skin / Q000378:metabolism / Q000503:physiopathology*
keywords: Acetic acid (PubChem CID: 176); Blue light; Calcein-AM (PubChem CID: 4126474); Cell viability; EDTA (Ethylenediaminetetraacetic acid) (PubChem CID: 6049); Ethanol (PubChem CID: 702); Ethidium homodimer (PubChem CID: 12328897); Fibroblasts; Formazan (PubChem CID: 16218671); Live/dead fluorescence; MTT; MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) (PubChem CID: 64965); Methods; Neutral red; Neutral red (PubChem CID: 11105); Phosphate-Buffered Saline (PubChem CID: 24978514); Phototherapy; Trypan blue; Trypan blue (PubChem CID: 9562061)