Seahorse xfp cell mito stress kit

Sphere-derived Panc185, PancA6L and Panc215 cells were plated in XF HS Miniplates (Seahorse Bioscience) at a cellular density of 5,000 cells/well. For OCR determination, cells were incubated in Seahorse XF DMEM media (103680, Agilent) supplemented with 2mM glutamine, 10mM glucose, and 1mM pyruvate for 1 h, prior to the measurements using the Seahorse XFp Cell Mito Stress Kit (103010, Agilent). After an OCR baseline measurement, the minimum oxygen consumption was determined adding 1.5µM oligomycin (O) and the maximal respiration rate was assessed by adding 1µM FCCP (F). At the end of the experiment the non-mitochondrial oxygen consumption was evaluated adding both 0.5µM rotenone (R) and antimycin (A). Experiments were run in a XF HS Mini analyzer (Seahorse Agilent), and raw data were normalized to total protein using BCA protein assay kit (Cat. no. 23225, Thermo Scientific).

Cell bioenergetics was evaluated in a Seahorse XFp analyzer (Agilent Technologies, Santa Clara, CA, USA). SH-SY5Y cells were plated on Seahorse XFp plates (55,000 cells/per well). After 24 h, cells were washed with XF DMEM medium followed by a 1 h incubation at 37 °C in a CO₂-free incubator. Each plate contained two wells without cells to serve as blank controls. After monitoring basal respiration, cells were sequentially treated with 1.5 μM oligomycin, 1 μM FCCP, and 0.5 μM antimycin A + 0.5 μM rotenone (Seahorse XFp Cell Mito Stress kit, from Agilent Technologies). Oxygen consumption rate (OCR) data were normalized to the total cell amount per well estimated by Janus Green staining [61 (link)]. Briefly, after bioenergetics analysis, cells were fixed in 4% paraformaldehyde (PFA) and incubated with 0.2% Janus Green B in PBS for 3 min at room temperature. The excess of dye was removed by dipping into cold water and gentle shaking, and the bound dye was dissolved in 0.5 N HCl (0.1 mL/well) and the optical density at 595 nm was evaluated. The metabolic parameters of the assay were calculated with the Seahorse Wave software. In all cases, 3 technical replicates were recorded in every experiment, and the experiments were carried out with 3 biological replicates.