Seahorse measurement of cellular respiration

Seahorse measurement of cellular respiration was performed using a Seahorse extracellular flux analyzer and XF Mito Stress kit (Agilent) according to the manufacturer’s protocol. In brief, 50,000 or 75,000 astrocytes were plated per well on a Matrigel coated XF 96-cell culture plate 3 days before the measurement and incubated in 37°C and 5% CO2 in a humidified incubator. Cells were cultured in 8 wells per group as technical replicates. On the day of measurement, cells were washed with XF cell Mito stress test assay medium containing Seahorse XF DMEM media (phenol red free), 10 mM glucose, 1 mM Pyruvate and 2 mM L-Glutamine. Cells were incubated in the assay medium for 1 h prior to the measurement in a CO2-free incubator at 37°C. During the measurement assay, inhibitors were loaded into cells at final concentration of 1.0 μM Oligomycin (Port A), 2 μM FCCP (Port B) and 0.5 μM Rotenone/antimycin A (Port C). Results of the measurement were subsequently analyzed using the Wave software (Agilent). Data was normalized by cell number as determined by Hoechst staining. OCR was presented as pmol/min per 100,000 cells.