Assessment of mitochondrial function is typically performed using one of three approaches. Protocol 1: After establishing baseline OCR, a compound of interest is injected. The effect on OCR and ECAR are then monitored over time. Protocol 2: After establishing baseline, the compound of interest is injected, OCR and ECAR are monitored for a predetermined amount of time, and then mitochondrial function is assessed. Protocol 3: Treatment with the compound of interest is completed outside the instrument. The cells are then washed free of the stressor, and mitochondrial function is directly assessed.

The effect of HNE on OCR and ECAR was assessed using a Protocol 1-style strategy. Panel A: After establishing baseline OCR in NRVM seeded to 75,000 cells/well, HNE was injected to a final concentration of 5 μM (open squares, solid line) or 20 μM (closed squares, dashed line). The effect on OCR was followed for an additional 4 h. Panel B: Same as Panel A, except ECAR was measured. Panel C: MES-13 were seeded at 30,000 cells/well, baseline OCR was established then HNE was injected at 5 μM (open squares, solid line) or 20 μM (closed squares, dashed line) and OCR was followed for 2 h. Panel D: Same as Panel E, except ECAR was measured. Panel E: After establishing baseline OCR in differentiated SH-SY5Y cells, HNE was injected to a final concentration of 0, 10, 15, or 20 μM. The effect on OCR was followed for an additional 2 h. Panel F: Same as Panel E, except ECAR was measured. Panel G: NRVM were exposed to 5, 10, or 20 μM HNE for 1 h. OCR and ECAR are plotted against each other from this single time point. Panel H: MES13 cells were exposed to 5, or 20 μM HNE for 1 h. OCR and ECAR are plotted against each other from this single time point. Panel I: Differentiated SH-SY5Y cells were exposed to 10, 15, or 20 μM HNE for 1 h. OCR and ECAR are plotted against each other from this single time point. All data shown are the mean ± sem. n≥3 per treatment group. Statistical significance is omitted for clarity. Where error bars are not visible, they are smaller than the data point symbol. Data in panels A, B and G are adapted from [].

The impact of HNE on mitochondrial function, protein modification, and HO-1 induction was examined following OCR measurements in RASMC. Panel A: The OCR of rat aortic smooth muscle cells was measured followed by acute exposure to 0, 0.5, and 5 μM HNE. OCR was then examined for the indicated time. Panel B: At the end of the experiment, cells were lysed in detergent-containing buffer (as described in the text), and the protein was separated by SDS-PAGE, followed by western blotting for protein-HNE adducts and HO-1. The PVDF membrane was then stripped of antibodies and stained with Amido Black (0.1% Naphthol Blue-Black in 40% methanol and 10% acetic acid). The PVDF was then destained with 40% methanol and 10% acetic acid to visualize the separated proteins. Panels C and D: HO-1 expression (Panel C) and protein-HNE adducts (Panel D) were quantified and normalized to Amido Black protein stain. n = 3 per group; *, p≤0.05 vs. cells not treated with HNE.

Panel A describes the approach to performing a Protocol 2-style experiment. Panel B: A typical profile of the mitochondrial function assay used to assess the acute effect of stressors on mitochondrial function. The example data is from control BAEC. After baseline OCR is established, the stressor of interest is injected and monitored for the desired amount of time. This allows the determination of the direct effect of the stressor on OCR. Then the mitochondrial function assay is performed as described. Individual parameters that can be calculated using this approach are indicated. Panel C: BAEC were seeded to 40,000 cells/well and allowed to incubate for 24 h. Basal OCR was then measured prior to injection of Deta NONOate to a final concentration of 250 or 500 μM. The cells were allowed to incubate for 1 h, then 3 additional measurements of OCR were made. This was followed by sequential injection of oligomycin (1 μg/ml), FCCP (1 μM), and antimycin A (10 μM). Panel D: From the data in Panel C, the reserve capacity of the mitochondrial can be calculated. Data shown in Panels C and D represent the mean ± sem. n≥3 per treatment group. *, p≤0.05 as compared to 0 μM Deta NONOate control. Panel C and D are adapted from Free Radical Biology and Medicine, v.48(7), Dranka BP et.al, Mitochondrial reserve capacity in endothelial cells: The impact of nitric oxide and reactive oxygen species, p. 905-14, Copyright (2010), with permission from Elsevier.

Employing a Protocol 2 strategy, the effect of HNE on mitochondrial function was examined in NRVM (Panels A and B), MES13 (Panels C and D), and SH-SY5Y (Panels E and F). Panel A: After establishing baseline, HNE was injected into NRVM cultures to a final concentration of 5, 10, or 20 μM. A final basal OCR measurement was taken 30 min later which was termed the post-HNE OCR. This was followed by the mitochondrial function assay as described. Panel B: Individual mitochondrial function parameters calculated from the data shown in Panel A. Panel C: Same as Panel A, except experiments were performed in SH-SY5Y cells. Panel D: Individual mitochondrial function parameters calculated from the data shown in Panel C. Panel E: Same as Panel A, except experiments were performed in MES-13 cells. Panel F: Individual mitochondrial function parameters calculated from the data shown in Panel E. All data shown are the mean ± sem. Where error bars are not visible, they are smaller than the data point symbol. n≥3 per treatment group. *, p≤0.05 as determined by two-tailed, unpaired t-test compared to the respective control measurement.

Using a Protocol 2 strategy, the effect of the redox-cycling compound DMNQ was examined in BAEC. After establishing baseline OCR, DMNQ was injected to a final concentration of 15 μM. Panel A depicts the effect on mitochondrial function. Basal OCR calculated from these data were then plotted as a function of DMNQ concentration (Panel B). From the data shown in Panel A, determinations of individual parameters of mitochondrial function can be made (Panel C). In some experiments, DPI (10 μM) was added at the time of DMNQ injection. DMNQ-stimulated OCR is shown in Panel D. All data shown are the mean ± sem. n=5 per treatment group. *, p≤0.05 as determined by two-tailed, unpaired t-test compared to the respective control measurement. Panels A and B are adapted from Free Radical Biology and Medicine, v.48(7), Dranka BP et.al, Mitochondrial reserve capacity in endothelial cells: The impact of nitric oxide and reactive oxygen species, p. 905-14, Copyright (2010), with permission from Elsevier.

Using a Protocol 3-style approach, the effect of oxidative stressors was tested in various cell lines pretreated for a period of time. In each case, the stressor was washed out prior to assessment of mitochondrial function. Panel A describes the approach to performing a Protocol 3-style experiment. Panel B: NRVM were seeded to 75,000 cells/well for 24 h. Some cells were then treated with a bolus addition of 50 μM H₂O₂ for 30 min. The cells were washed, and changed to assay media for 1 h prior to assessment of mitochondrial function using 1μg/ml oligomycin, 1μM FCCP, and 10 μM antimycin A. Panel C: Individual mitochondrial function parameters calculated from data in Panel B. Data shown are the mean ± sem. n≥3 per treatment group. Panel D: MDA-MB231 cells were treated for 30 min with 10 μM 15d-PGJ₂ (15d), mito-15d-PGJ₂ (Mito-15d) or mito-PGE₂ and mitochondrial function was assessed as described. After baseline was established, sequential injection of oligomycin (0.3 μg/ml), FCCP (1 μM) and antimycin A (10 μM) was used to determine mitochondrial function parameters. *, p≤0.05 as compared to control cells. #, p≤0.05 as compared to untargeted 15d-PGJ₂ treated cells. Results represent means ± sem, n=5.