m was removed, fresh medium was added, and the cells were treated with different concentrations of clotrimazole for 24 h. After this incubation, the medium was removed, and the ATP Lite kit reagents were added. This system is based on the production of light by luciferase as it consumes ATP 14 / 20 Anticancer Effects of Nanomicellar Clotrimazole and D-luciferin. The luminescence is proportional to the concentration of cellular ATP and was analyzed with a VICTOR3 multilabel reader . Cell viability MCF-7, MCF10-A and C2C12 cells were seeded in 96-well plates and grown to confluence. Then, the medium was removed, fresh medium was added, and the cells were returned to the incubator in the presence of different concentrations of clotrimazole. The micelle BCTC components, DMSO and Tween 80 were used as negative controls. After 24 h, the medium was removed, and the amount of leaked lactate dehydrogenase was evaluated by monitoring the reduction of NAD+ to NADH via the absorbance at 340 nm in a VICTOR3 multilabel reader . Succinate dehydrogenase activity Cells were seeded in 96-well plates and grown to confluence. PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19736355 Then, the medium was removed, fresh medium was added, and the cells were treated with different concentrations of clotrimazole for 24 h. After this incubation, the medium was removed, and the PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19735252 cells were snap-frozen at -80C for 1 h and then incubated for 10 min with 12.3 mM malonate, a competitive inhibitor of succinate dehydrogenase, and 10 mM potassium phosphate buffer. The cells were then incubated in the dark at 25C in a reaction buffer containing 12.3 mM diethyl succinate, 0.2 mM 1-methoxy 5-methylphenazinium methyl sulfate, 1.2 mM nitro-blue-tetrazolium and 50 mM Tris HCl, pH 7.6. The activity of SDH was determined spectrophotometrically using NBT, which turns purple when it accepts electrons, as an artificial electron acceptor and succinate as the substrate. The purple color is directly proportional to enzyme activity and was measured in a VICTOR3 multilabel reader . Analysis by 
scanning electron microscopy Cells were seeded in 24-well plates with glass coverslips on the bottom and grown to confluence. Then, the medium was removed, fresh medium was added, and the cells were treated with different concentrations of clotrimazole for 24 h. The micelle components, DMSO and Tween 80 were used as negative controls. After this incubation, the coverslips were removed and their adherent cells were washed twice with PBS and fixed in a 0.1 M cacodylate-NaOH buffer containing 2.5% glutaraldehyde, 5 mM CaCl2 and 3.7% sucrose for 1 h. After that, cells were further fixed for additional 1 h in a 0.1 M cacodylateNaOH buffer containing 1% OsO4, 0.8% K4Fe6 and 5 mM CaCl2. Then, the cells were washed in 0.1 M cacodylate-NaOH buffer, dehydrated in graded ethanol, and dried with CO2 stream. Dried samples were further adhered to 20 nm gold layer-coated scanning electron microscopy stubs using a sputtering device. JEOL JSM 5310 scanning electron microscope operating at 25 kV was used to observe the cells. Ultrastructural analysis by transmission electron microscopy Cells were seeded in 24-well plates with glass coverslips on the bottom and grown to confluence. Then, the medium was removed, fresh medium was added, and the cells were treated with different concentrations of clotrimazole for 24 h. The micelle components, DMSO and Tween 80 were used as negative controls. After this incubation, the coverslips were removed and their adherent cells were washed tw
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