Seahorse xfp analyser

Transfected cells were cultured 24 h in normoxic (21% O₂) condition followed by 24 h of normoxic or hypoxic (1% O₂) condition, and the secreted lactate concentration was measured from cell culture medium by using l-Lactate colorimetric assay (Abcam). Cells were stained for nuclear stain Hoechst 33342, and lactate concentration was normalized to cell count.
Cell culture medium pH was measured from transfected cells cultured 24 h in normoxic condition followed by 24 h of normoxic or hypoxic condition. For pH measurements, the cells were stained for nuclear stain Hoechst 33342 and hydronium ion [H₃O⁺] concentration was normalized to cell count and converted to pH.
Transfected cells were seeded (1 × 10⁴) on the 8-well Seahorse XFp Miniplate (Agilent Technologies) in triplicates. Prior analysis, cells were incubated for 1 h in XF Base medium (Agilent Technologies) supplemented with glucose, glutamine and pyruvate in a non-CO₂ incubator at 37 °C. Seahorse XFp Analyser (Agilent Technologies) and Seahorse XF Cell Mito Stress Test Kit was used for determining the oxygen consumption rate (OCR) and glycolysis function by using the manufacturer’s protocol.

Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in live cells were measured using a Seahorse XFp analyser (Agilent) according to the manufacturer’s instructions. Cells were seeded into Seahorse XFp 96 well microplates at an optimised density and 24 h hours later, cells were washed and incubated with Seahorse XF Cell Energy Phenotype Assay Media.FCCP titration assays were performed to determine the optimum concentration for the Mito Stress Test use, which was performed using the Seahorse XFp Cell Mito Stress Test kit (Agilent). Cells were washed and kept with prepared assay media and incubated without CO₂ for 45 min at 37℃. The standard Cell Mito Stress Test protocol was performed without further modification to allow OCR and ECAR to be calculated.

Metabolic analyses of U87 cells were preformed using the Seahorse XFp analyser (Agilent; North Billerica, MA, USA). Cells (22,000/well) were seeded in an XFp 96-well plate, and then treated with vehicle or Ki8751(2.5 µM). After incubation for 24 h, cells were used for Mito Stress assay. Cells were first washed and preincubated for one hour with Seahorse XF DMEM medium (pH7.4) (Agilent) supplemented with 1 mM sodium pyruvate, 10 mM glucose, and 2 mM L-glutamine (Sigma). Oxygen consumption rate (OCR) was analyzed at basal conditions and after sequential injections of oligomycin (1 µM), carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP; 1 µM), and antimycin/rotenone (0.5 µM). All metabolic assays were normalized to the total protein content.