Xf96 well cell culture plates

The extracellular acidification rate in real time (ECAR) was determined using the Seahorse Extracellular Flux Analyzer (XF96, Seahorse Bioscience, Billerica, MA, USA). H295R cancer cells were seeded into XF96-well cell culture plates (Seahorse Bioscience, Billerica, MA, USA), and incubated overnight at 37 °C in a 5% CO₂ humidified atmosphere. After 48 h, cells were treated with curcumin (10, 20 μM) for 16 h. At the end of treatment, cells were processed as previously described [3 (link)]. The obtained ECAR values were normalized to the protein content within the individual wells.

The extracellular acidification rate in real time (ECAR) was determined using the Seahorse Extracellular Flux Analyzer (XF96) (Agilent). Adrenocortical cancer cells (H295R, SW13, MUC-1) and H295R clones (shCTR, shERRα−/−, ERRα+/+) were seeded into XF96-well cell culture plates (Seahorse Bioscience, MA, USA), and incubated overnight at 37 °C in a 5% CO₂ humidified atmosphere. After 48 h, cells were treated with XCT790 (1, 5, 10 μM) for 18 h. At the end of treatment, cells were washed in a specific buffer (XF medium, pH 7.4) for the determination of metabolic flows added with 2 mM of L-glutamine. The cells were then maintained for 1 h in 175 μL of XF medium at 37 °C, in an incubator without CO₂. During the incubation time, the XF buffer solution (25 µL) containing glucose (10 mM) oligomycin (1μM), 2-deoxy-D-glucose (50 mM) was added into the injection ports. ECAR measurements were normalized to protein content within the individual wells. The dataset was analyzed by XFe-96 software (Agilent).

Cells were plated in a XF96-well cell culture plates (Agilent, Santa Clara, CA, USA) at a density of 20,000 cells per well. The Mitochondrial Stress Test and Real-Time ATP Rate Assay were performed independently in a Seahorse XF Analyzer (Agilent Technologies, Santa Clara, CA), as recommended by the vendor. Briefly, H1299 cells were incubated with unbuffered Seahorse XF Base medium supplemented with 10 mM glucose, 2 mM L-glutamine, 1 mM sodium pyruvate and adjusted to pH 7.4. Cells were incubated for 1 h in a non-CO₂ incubator prior to sequential injections of Mitochondrial Stress or ATP Test compounds. For the Mitochondrial Stress Test, XFe96 cartridge ports were loaded with oligomycin (1 µM), FCCP (2 µM) and a combination of rotenone and antimycin-A (both at 0.5 µM), all diluted in supplemented Seahorse XF Base medium. For the ATP Rate Assay, FCCP was excluded. Results obtained were normalised for total protein content, using a standard BCA assay and normalised data analysed using the Seahorse XF Mitochondrial Stress and ATP Test generator from Agilent.

TNBC cells were continuously treated by TNFα + IL-1β/vehicle. Oxidative phosphorylation and glycolytic rates of the cells were assessed using a Seahorse XFe96 Analyzer (Agilent Technologies Inc., Santa Clara, CA, USA). To this end, TNBC cells were cultured in XF-96-well cell culture plates (Agilent). After 24 h the media were replaced by fresh complete media with TNFα + IL-1β/vehicle for 48 h. For the mito stress test, oxygen consumption rate (OCR) measurements were taken at basal rate followed by injections of oligomycin (1 μM; #O4876, Sigma-Aldrich), carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP; 1 μM; #C2920, Sigma-Aldrich) and a mixture of rotenone (Rot; 0.5 μM; #R8875, Sigma-Aldrich) + antimycin A (AA; 0.5 μM; #A8674, Sigma-Aldrich). For the glycolytic rate assay, extracellular acidification rate (ECAR) measurements were taken at basal rate followed by injections of mixture of Rot (0.5 μM; #ab143145, Abcam, Cambridge, MA, USA) +AA (as above), and then 2-Deoxy-D-Glucose (2-DG; 50 mM; #ab142242, Abcam).
Following analysis, the cells were fixed with 4% paraformaldehyde and stained with Hoechst (#B2261, Sigma-Aldrich). Each well was photographed at different regions and metabolic signals were normalized to cell numbers.