A simple method to measure cell viability in proliferation and cytotoxicity assays.
Resazurin dye has been broadly used as indicator of cell viability in several types of assays for evaluation of the biocompatibility of medical and dental materials. Mitochondrial enzymes, as carriers of diaphorase activities, are probably responsible for the transference of electrons from NADPH + H+ to resazurin, which is reduced to resorufin. The level of reduction can be quantified by spectrophotometers since resazurin exhibits an absorption peak at 600 etam and resorufin at 570 etam wavelengths. However, the requirement of a spectrophotometer and specific filters for the quantification could be a barrier to many laboratories. Digital cameras containing red, green and blue filters, which allow the capture of red (600 to 700 etam) and green (500 to 600 etam) light wavelengths in ranges bordering on resazurin and resorufin absorption bands, could be used as an alternative method for the assessment of resazurin and resorufin concentrations. Thus, our aim was to develop a simple, cheap and precise method based on a digital CCD camera to measure the reduction of resazurin. We compared the capability of the CCD-based method to distinguish different concentrations of L929 and normal Human buccal fibroblast cell lines with that of a conventional microplate reader. The correlation was analyzed through the Pearson coefficient. The results showed a strong association between the measurements of the method developed here and those made with the microplate reader (r(2) = 0.996; p < 0.01) and with the cellular concentrations (r(2) = 0.965; p < 0.01). We concluded that the developed Colorimetric Quantification System based on CCD Images allowed rapid assessment of the cultured cell concentrations with simple equipment at a reduced cost.
Borra
Ricardo Carneiro
RC
Ibirapuera University, São Paulo, SP, Brazil. rcborra@gmail.com
Lotufo
Mônica Andrade
MA
Gagioti
Sonia Maria
SM
Barros
Fabiana de Mesquita
Fde M
Andrade
Priscila Maria
PM
D002151:Calorimetry / Q000379:methods; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects*; D002478:Cells, Cultured / Q000187:drug effects; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects*; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism / Q000633:toxicity*; D009061:Mouth Mucosa / Q000166:cytology; D010078:Oxazines / Q000378:metabolism / Q000633:toxicity*; D010781:Photography / Q000295:instrumentation / Q000379:methods; D018675:Toxicity Tests / Q000379:methods*; D014966:Xanthenes / Q000378:metabolism / Q000633:toxicity*
D007202:Indicators and Reagents; D010078:Oxazines; D014966:Xanthenes; C005843:resazurin; C014180:resorufin