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Mitochondrial Respiratory Profile of ovarian cancer cell lines.

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posted on 23.05.2014, 03:24 by Usawadee Dier, Dong-Hui Shin, L. P. Madhubhani P. Hemachandra, Larissa M. Uusitalo, Nadine Hempel

A. Oxygen Consumption Rate (OCR) measurements were obtained over time (min) using an extracellular flux analyzer (Seahorse Bioscience). The mitochondrial stress test was used to obtain bioenergetics parameters, by adding the ATP synthase inhibitor Oligomycin A (O, 1 µM), to derive ATP-linked OCR, FCCP (F, 750 nM) to uncouple the mitochondria for maximal OCR, and Antimycin A (A, 1 µM). B. Basal mitochondrial OCR of ovarian cancer cell lines was derived by subtracting non-mitochondrial OCR (remaining OCR after Antimycin A addition). C. ATP-linked OCR was derived as the difference between basal and Antimycin A inhibited OCR. D. OCR attributed to proton leak was calculated as the difference between OCR following Oligomycin A inhibition and OCR following Antimycin A inhibition. E. Maximum OCR was stimulated by FCCP addition. F. The respiratory reserve capacity was calculated as the difference between maximal and basal OCR. Graphs B-F represent data from one replicate experiment (n = 5, ANOVA, Tukey's post test *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 statistically significant compared to NOSE007; #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001 statistically significant compared to OVCA420). OCCC cell lines are highlighted in black and a normal epithelial cell line in white. G. Average Respiratory StateApparent was calculated from 3–4 replicative experiments using: 4−(Basal OCR−Oligo OCR)/(FCCP OCR−Oligo OCR).