Figure 1.
Schematic flowchart for the isolated mitochondria assay using the Seahorse XF24 Analyzer.
Mitochondria are diluted into 1X MAS containing the substrate of choice. Initial conditions refer to any additives or compounds present at 1X at the start of the assay in addition to the substrate (e.g. drug candidate, etc.).
Table 1.
Mix and Measure Cycle Times.
Figure 2.
Optimization of isolated mitochondria XF assays.
2A–B, Determination of optimal µg amount of mitochondria/well. 1.25–40 µg/well of mouse liver mitochondria were attached to a V7 polystyrene XF24 plate and the coupling experiment was performed as described in Methods in the presence of succinate/rotenone. Blue vertical lines denote injections of indicated compounds. 2A shows OCR for 1.25–40 µg samples. 2B shows the absolute O2 tension (in mm Hg) in the microchamber for 1.25–40 µg samples. Note that samples at 10 µg and above show unstable State 3 rates for OCR and depletion of O2 in the microchamber in panels A and B, respectively. Lettering within data points indicates the group identification number.
Figure 3.
Characterization of mitochondrial activity.
3A, Titration of ADP using 5 µg mouse liver mitochondria/well. ADP (0–4 mM) was injected via port A to initiate State 3 respiration and the measurement time was extended to 6 minutes. Note that 2–4 mM ADP is sufficient to maintain a relatively stable State 3 respiration rate for the duration of the measurement period, while lower concentrations show exhaustion of ADP and transition to State 4 respiration. 3B, Alkalinization of the media during phosphorylating respiration (note that unlike the OCR tracings, this data reports absolute pH rather than a rate of change in pH).
Figure 4.
Isolated mitochondria remain attached to the plate for the duration of the experiment.
A, The coupling experiment was performed using 5 µg mouse liver mitochondria per well as described in Methods, however, State 3 respiration was allowed to proceed for multiple measurement periods (average OCR per measurement period shown). Note that State 3 respiration does not diminish over multiple mixing and measuring periods, indicating that the mitochondria remain attached to the well for the duration of the assay. B, State 4o rates using different plate coatings in the presence and absence of 0.2% BSA. No significant differences in State 4o rates were observed among different plate coatings [none, polyethyleneimine (PEI), and Cell-Tak®]. Note that the absence of BSA resulted in elevated rates of State 4o respiration, indicative of respiratory uncoupling. C–D, Isolated mitochondria adhered to the XF24 plate as imaged by phase contrast microscopy at 20X magnification before (4C) and after the XF assay (4D).
Figure 5.
Comparison of Clark electrode and XF technology shows comparable respiration data between the methods.
Mitochondria isolated from rat heart and mouse liver were used in parallel coupling experiments using either a Hansatech or Rank Clark type electrode or the XF24. Assays were performed as described in Methods for each platform, respectively. Comparison of Basal, State 3, State 4o and State 3u rates between the Hansatech and XF with rat heart using or glutamate/malate as substrate (5A) or succinate/rotenone (5B), respectively. Comparison of Basal, State 3, State 4o and State 3u rates between the Rank and XF with mouse liver mitochondria using succinate/rotenone (5C). Data are expressed as mean ± SD from 3 separate experiments in Fig 5A and B, and mean ± SD from 4 experiments in 5C. The high SD in 5C owes to higher rates obtained with one of the four mouse liver preps, rather than variation between methodologies on a given day. Data were analyzed using a two-factor ANOVA with repeated measures on one factor. An interaction was detected only in the data of Fig. 5A, and post-hoc paired comparisons detected lower rates in the XF24 of State 3 and 3u, and a higher rate of State 4o respiration with rat heart mitochondria oxidizing glutamate and malate (p<0.05).
Figure 6.
Using the Coupling and Electron Flow assays in tandem to elucidate mechanistic activity of test agents.
Coupling (A, C) and electron flow experiments (B, D) were performed as described in Methods. Initial conditions are as follows (with final concentrations listed): A–B, Controls (no additives) or 20 mM sodium azide or 4 µM antimycin-A; C–D, Controls (no additives) or 10 mM malonate or 2.5 µg/ml oligomycin or 2 µM rotenone. See text for further explanation of results.
Figure 7.
In 7A, intra-assay reproducibility is demonstrated. Assays used 3–5 replicate wells and well-to-well variation within electron flow and coupling assays shows coefficients of variation (CV) <17% in all measurements (except where OCR has been reduced to minimal levels with rotenone or antimycin-A). In 7B, inter-assay reproducibility is demonstrated. Four separate uncoupling experiments from four different preparations of mouse liver mitochondria were averaged to illustrate reproducibility of the assay over multiple days/preparations and shows CVs <20%. The corresponding table indicates the means, standard deviations and% CV, average RCR values are given in the text.
Figure 8.
Effects of Phosphatase Inhibitor (PPI) treatment on rat heart mitochondrial respiration.
Mitochondria were treated with a cocktail of phosphatase inhibitors during the isolation procedure as described in the Methods. Respiratory States 3, 4o and 3u were measured in the presence (+PPI) or absence (-PPI) of phosphatase inhibitor treatment in the presence of either succinate/rotenone (3 µg/well) or glutamate/malate (6 µg/well) as oxidizable substrates, and rates are expressed per µg mitochondrial protein.