Xf sensor cartridge

The role of mitochondrial respiration was assessed via the addition of different regulators, and the OCR was analyzed according to previous studies with some modifications [15 (link),16 ]. Two days before the experiment, hMSCs were seeded at a density of 1.4 × 10⁴ cells per Seahorse culture microplate well on gelatin-coated plates. The Seahorse Analyzer was warmed up 1 d in advance.
The assay medium was then prepared by supplementing the basal medium with 2 mM glutamine, 1 mM sodium pyruvate, and 10 mM glucose. The cell culture microplate was incubated in a CO₂-free incubator at 37 °C for 1 h and washed twice with the assay medium before incubation. The Seahorse XF sensor cartridge was precalibrated and then filled with mitochondrial respiration regulators rotenone/antimycin A (0.5 μM), oligomycin (1 μM), and FCCP (2 μM). The software “Wave” was used to conduct and analyze the experiment, and the values of OCR were finally normalized with cell number per well.

Metabolic measurements were done in real-time, non-invasively, using a Seahorse XF96 analyser, which measured the OCR and the ECAR under basal conditions in siDAPK2- or siNS-transfected cells. A cell titration assay was used to determine a suitable cell plating density for both A549 and U2OS cells. For the experiment proper, 0.5 × 10⁶ cells were plated per 10 cm dish and reverse transfected on day 1 as previously described. Forty-eight hours post transfection, cells were re-plated into the XF96 microplates (Seahorse Bioscience; 4 × 10⁴ cells per 100 μl per well) and incubated overnight at 37 °C. The XF calibration solution (Seahorse Bioscience) was added into the XF sensor cartridge (Seahorse Bioscience) and was also incubated at 37 °C overnight but without CO₂. The next day, prior to the assay, complete DMEM was replaced with an XF Assay Medium Modified DMEM (Seahorse Bioscience) (1 g/ml glucose, pH 7.4) and cells were incubated at 37 °C for 1 h without CO₂. Analyses were performed according to the manufacturer's instructions using eight measurements that the instrument recorded for OCR (nmoles/min) and ECAR (mpH/min) pertaining to each well. Results were analysed using the provided XFe Wave software (Seahorse Bioscience).

The metabolic alternations of cultured cells were monitored using an XF96 metabolic flux analyzer (Seahorse Biosciences, Billerica, MA, USA) according to the manufacturer’s instructions. The ROBO1 or SLIT2 siRNA-transfected and negative control siRNA-transfected cells were plated in wells of a model XF96 96-well plate (Seahorse Biosciences; 3 × 10⁴ cells in the 80 μL added to each well) and incubated overnight at 37 °C. The XF calibration solution (Seahorse Biosciences) was added to the XF sensor cartridge (Seahorse Biosciences) and was also incubated at 37 °C overnight but without CO₂. The next day, prior to the assay the complete medium was replaced with XF assay modified DMEM medium (1 g/mL glucose, pH 7.4; Seahorse Biosciences) and the cells were incubated at 37 °C for 1 h without CO₂. The real-time glycolytic rate (ECAR) was assessed by the sequential injection of 10 mM glucose, 1 mM oligomycin (Sigma-Aldrich), and 80 mM 2-deoxyglucose (D8375; Sigma-Aldrich). The OCR was assessed by the sequential injection of 1 mM oligomycin, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (C2920; Sigma-Aldrich, C2920), 2 mM antimycin A, and 2 mM rotenone (Sigma-Aldrich). The instrument recorded 12 measurements for ECAR (nmoles/min) and OCR (mpH/min) in each well. The results were analyzed using the XFe Wave software (Seahorse Biosciences).