Mitochondrial activity was evaluated using resazurin sodium (10-oxide of 7-hydroxy-3hydro-phenoxacin-3-one sodium salt, Sigma). The RAW 264.7 cells were seeded in 24-well plates at a density of 2.6 × 10 4 cells/cm 2 , and after reaching confluence (at 3 days postseeding) were exposed over 4, 24 and 48 h to different concentrations of fluoride (2.5, 5, 10, 20, 50, 65 and 75 mg/L, equivalent to 0.13, 0.26, 0.53, 1.1, 2.6, 3.7 and 4.1 mM NaF, respectively) . The fluoride standards were prepared in minimum essential medium with Earle's salts (MEM, PAA) supplemented with 100 U/mL of penicillin, 0.1 mg/mL of streptomycin, 0.0025 mg/mL of amphotericin B, 1 mM of sodium pyruvate and 10 mM of HEPES.
Following exposure, the medium was removed and the cultures were washed twice with PBS (500 µL). Then 500 µL of resazurin solution (10 µg/mL in supplemented MEM) were added, followed by incubation for 1 h at 37ºC in an atmosphere with 95% relative humidity and a CO 2 flow of 5%. The decrease in resazurin was determined by spectrophotometry performing readings at 570 and 600 nm (PowerWave HT Microplate Scanning Spectrophotometer, Bio-Tek instruments, USA). The results were expressed as percentages with respect to the absorbance of cells not treated with fluoride.
Following exposure, the medium was removed and the cultures were washed twice with PBS (500 µL). Then 500 µL of resazurin solution (10 µg/mL in supplemented MEM) were added, followed by incubation for 1 h at 37ºC in an atmosphere with 95% relative humidity and a CO 2 flow of 5%. The decrease in resazurin was determined by spectrophotometry performing readings at 570 and 600 nm (PowerWave HT Microplate Scanning Spectrophotometer, Bio-Tek instruments, USA). The results were expressed as percentages with respect to the absorbance of cells not treated with fluoride.