Abstract
Extracellular flux (XF) analysis has become a mainstream method for measuring mitochondrial function in cells and tissues. Although this technique is commonly used to measure bioenergetics in intact cells, we outline here a detailed XF protocol for measuring respiration in permeabilized cells. Cells are permeabilized using saponin (SAP), digitonin (DIG) or recombinant perfringolysin O (rPFO) (XF-plasma membrane permeabilizer (PMP) reagent), and they are provided with specific substrates to measure complex I– or complex II–mediated respiratory activity, complex III+IV respiratory activity or complex IV activity. Medium- and long-chain acylcarnitines or glutamine may also be provided for measuring fatty acid (FA) oxidation or glutamine oxidation, respectively. This protocol uses a minimal number of cells compared with other protocols and does not require isolation of mitochondria. The results are highly reproducible, and mitochondria remain well coupled. Collectively, this protocol provides comprehensive and detailed information regarding mitochondrial activity and efficiency, and, after preparative steps, it takes 6–8 h to complete.
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Acknowledgements
We acknowledge support from the US National Institutes of Health (grant nos. GM103492 and HL078825).
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J.K.S., A.A.G. and B.G.H. were involved in all parts of manuscript preparation.
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Integrated supplementary information
Supplementary Figure 1 Experimental design template.
Experimental template designed to assist in designing and executing XF experiments.
Supplementary Figure 2 The presence of BSA in MAS buffer preserves mitochondrial coupling.
(a) XF analyzer trace of OCR over time: Smooth muscle cells were pre-exposed to 3 nM rPFO in MAS buffer in the presence or absence of fatty acid-free BSA. After 5 min pre-exposure to rPFO, cells were loaded into the XF analyzer and OCR recorded. rPFO; recombinant perfringolysin (XF PMP). (b) RCR values in the absence or presence of BSA in MAS buffer. These data show that BSA is required to optimize mitochondrial coupling. Such preservation of mitochondrial coupling is typically observed when assays are performed in MAS buffer containing 2-4 mg/ml BSA.
Supplementary Figure 3 Comparison of rPFO and saponin for use in the permeabilized cell assay.
(a) XF analyzer traces of OCR in cells exposed to SAP/succ/rot/ADP or to rPFO/succ/rot/ADP. Cells were either pretreated with permeabilizer immediately before inserting plate into the XF24 analyzer (closed circles and triangles) and then given succ/rot/ADP, or media on cells was changed to MAS buffer in the absence of permeabilizing agent and then permeabilizer/succinate/rotenone/ADP were co-injected (opened circles and triangles); (b) States 3 and 4 OCR values in rPFO- and saponin-permeabilized cells. Values were calculated from cells to which permeabilizer was co-injected with substrates (i.e., opened circles and triangles); (c) RCR values calculated from (b). These results show that, in smooth muscle cells, rPFO (PMP reagent) and saponin work equally well in permeabilized cell assays. Furthermore, these data suggest that permeabilization before insertion of plate into the XF or during the XF assay works equally well. However, results may be highly dependent on the cell type. The activity of mitochondria in some cell types may be negatively affected by detergent-based permeabilizers such as saponin and the timing of permeabilization could prove critical. Therefore, in untested cell lines, the choice of permeabilizer and the timing of permeabilizer addition would need to be determined empirically.
Supplementary information
Supplementary Figure 1
Experimental design template. (PDF 51 kb)
Supplementary Figure 2
The presence of BSA in MAS buffer preserves mitochondrial coupling. (PDF 32 kb)
Supplementary Figure 3
Comparison of rPFO and saponin for use in the permeabilized cell assay. (PDF 95 kb)
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Salabei, J., Gibb, A. & Hill, B. Comprehensive measurement of respiratory activity in permeabilized cells using extracellular flux analysis. Nat Protoc 9, 421–438 (2014). https://doi.org/10.1038/nprot.2014.018
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DOI: https://doi.org/10.1038/nprot.2014.018
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