Endothelial cells were seeded at 20 000 cells/well on a Seahorse 96-well XF Cell Culture Microplate as detailed by the manufacturer (Seashore Bioscience/Agilent Technologies, Santa Clara, California, USA) and allowed to adhere overnight in complete EBM-2 (EBM-2 basal media plus EBM-2 SingleQuots, Lonza, Basel, Switzerland). The following day cells were rinsed with 1× PBS and 50% control or SLE patient serum was added to wells and allowed to incubate for 24 hours (six samples per group with five replicates per patient sample). The Seahorse XF Analyzer (Seashore Bioscience/Agilent Technologies) was used to determine basal oxygen consumption rate (OCR). Four basal rate measurements were followed by four measurement cycles following each injection (1 µM oligomycin, 1 µM carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone and 2 µM AA rotenone). Consumption rates were calculated as previously described.33 (link) The bioenergetic health index (BHI) was calculated using the following formula: BHI= (ATP-linked × Reserve Capcity)/(Proton Leak × Non-mitochondrial OCR).34
Protocol detail
Assessing Mitochondrial Bioenergetics in SLE
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Bioenergetic
Carbonyl cyanide
Cell culture
Cells
Endothelial cells
Mitochondrial
Oligomycin
Oxygen consumption
Patient
Phenylhydrazone
Proton
Rotenone
Seahorse
Serum
Corresponding Organization : Ralph H. Johnson VA Medical Center
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Variable analysis
independent variables
- Serum (control or SLE patient)
- Basal oxygen consumption rate (OCR)
- ATP-linked OCR
- Reserve capacity
- Proton leak
- Non-mitochondrial OCR
- Bioenergetic health index (BHI)
- Cell type (endothelial cells)
- Cell seeding density (20,000 cells/well)
- Cell culture media (complete EBM-2)
- Cell incubation time (24 hours)
- Measurement cycles (4 basal rate measurements, 4 measurement cycles after each injection)
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