OCR was measured using the Seahorse XF96 analyzer (Seahorse Bioscience,
Billerica, MD). Primary cells were suspended in XF Assay Medium supplemented
with 25 mM glucose, 1 mM pyruvate and PGF cocktail. 60,000-100,000 cells were
seeded per well of a Seahorse XF96 cell culture plate (35 μl volume)
pre-coated with Cell-tak (Fisher Scientific). Cells were left to adhere for a
minimum of 30 minutes (min) in a CO2-free incubator at 37°C, after which
140 µl of XF Assay Medium were added into each well. The plate was left
equilibrating for 10 min in the CO2-free incubator before being transferred to
the Seahorse XF96 analyzer. Measurement of OCR was done at baseline and
following sequential injections of a) oligomycin (1 µM), an ATP synthase
inhibitor b) FCCP (1.6 µM), a mitochondrial uncoupler c) antimycin A (1
µM) and rotenone (1 µM; all Sigma-Aldrich) a complex III and
complex I inhibitor respectively. This enabled us to measure the OCR coupled to
ATP production, as well as the maximal and the mitochondrial OCR respectively.
OCRs were normalized by cell number.
Billerica, MD). Primary cells were suspended in XF Assay Medium supplemented
with 25 mM glucose, 1 mM pyruvate and PGF cocktail. 60,000-100,000 cells were
seeded per well of a Seahorse XF96 cell culture plate (35 μl volume)
pre-coated with Cell-tak (Fisher Scientific). Cells were left to adhere for a
minimum of 30 minutes (min) in a CO2-free incubator at 37°C, after which
140 µl of XF Assay Medium were added into each well. The plate was left
equilibrating for 10 min in the CO2-free incubator before being transferred to
the Seahorse XF96 analyzer. Measurement of OCR was done at baseline and
following sequential injections of a) oligomycin (1 µM), an ATP synthase
inhibitor b) FCCP (1.6 µM), a mitochondrial uncoupler c) antimycin A (1
µM) and rotenone (1 µM; all Sigma-Aldrich) a complex III and
complex I inhibitor respectively. This enabled us to measure the OCR coupled to
ATP production, as well as the maximal and the mitochondrial OCR respectively.
OCRs were normalized by cell number.