Xf cell mitochondrial stress test

Seahorse XF Cell Mitochondrial Stress Test (Agilent) was carried out as recommended based on the manufacturer’s protocol. To prepare cell samples for analysis, 10⁵ cells per well were seeded for overnight incubation in XF96 tissue culture plates supplemented with 5% CO₂. Cells were then washed twice with 200 µL of XF base medium (pre-warmed) supplemented with 2 mM glutamine, 25 mM glucose and 1 mM sodium pyruvate pH 7.4, followed by 1 h incubation in 150 µl of XF base medium plus 25 µl growth medium (175 µl final) at 37 °C without CO₂ prior to analysis. To perform the Seahorse XF Cell Mitochondrial Stress Test, the following pre-warmed reagents in the amount of 25 µl each were loaded into injector ports of the sensor cartridge calibrated on the XF96 analyzer (Seahorse Bioscience, Billerica, MA, USA): oligomycin to port A (0.8 µM final), FCCP to port B (2.5 µM), and antimycin A to port C (1 µM final). OCR was first measured under basal conditions, and additional measurements were taken after the sequential addition of oligomycin, FCCP, and antimycin A. The measurements were normalized to cell numbers based on MTT cell proliferation assays.

The mitochondrial oxygen consumption rate (OCR) of NHU cells in the presence and absence of 3 mmol/L ketamine was assessed using an XFp Analyzer to perform an XF Cell Mitochondrial Stress Test, according to the manufacturer's instructions (Seahorse Bioscience, Copenhagen, Denmark), as detailed.¹¹ (link) Briefly, NHU cells were seeded at 1 × 10⁴ cells per well and cultured for 48 hours to near confluence, when cultures were exposed to medium containing 3 mmol/L ketamine and maintained for a further 48 hours. Before the assay, cultures were washed with, and then incubated in, XF Assay modified Dulbecco's modified Eagle's medium (unbuffered; Seahorse Bioscience) containing 5 mmol/L l-glutamine, 5 mmol/L sodium pyruvate, and 6 mmol/L glucose. The XFp Analyzer performed fluorimetric detection of oxygen consumption by sequentially adding and mixing 1 μmol/L oligomycin, 1 μmol/L carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), and 0.5 μmol/L antimycin A with 0.5 μmol/L rotenone at defined time points. Values were normalized to total cellular protein derived from a bicinchoninic acid assay (Pierce, supplied by Thermo Fisher Scientific), and mitochondrial respiration was quantified by normalization to OCR values in the presence of antimycin A and rotenone, as described elsewhere.¹¹ (link)