Xfe24 microplate

A549-13S, A549-12S and A549-EV cells were seeded on XFe24 microplates (Agilent, Santa Clara, CA, USA) at a density of 1 × 10⁵ cells/mL for 24 h. Following this, cells were washed in D-PBS (Wisent Inc) and incubated in Seahorse XF base medium (DMEM-based with no bicarbonate, glucose, or pyruvate; Agilent) supplemented with 2 mM L-glutamine at 37 °C in a CO₂-free incubator for 60 min. The microplate was transferred to a Seahorse XFe24 Analyzer (Agilent) to measure the extracellular acidification rate (ECAR). Basal ECAR was recorded for 3 measurement cycles before injection of glycolytic stress test compounds. Measurements were taken during sequential cycles of exposure to 10 mM glucose (3 measurement cycles), 1.5 μg/mL oligomycin (3 measurement cycles), and 50 mM 2-deoxyglucose (2-DG) (3 measurement cycles). The ECAR was normalized to the third measurement prior to injection of the first compound.

Expanded NK cells were seeded at a density of 400,000 cells per 100 ul in a well of a 24-well cell culture XFe24 microplates (Agilent Technologies) pre-coated with poly-d-lysine (Sigma). Cells were treated with obese or non-obese ACM or TCM for 24 h. Prior to the commencement of the assay microplates were centrifuged at 1300 rpm for 3 min to allow for adherence. Media was removed and cells were washed with unbuffered Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10 mM of glucose and 10 mM of sodium pyruvate, (pH 7.4) and incubated for one hour at 37 °C in a CO₂-free incubator. The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using a Seahorse XFe24 Extracellular Flux Analyser (Agilent Technologies).
Measurements were normalised to cell number using the crystal violet assay. Cells were fixed with 1% glutaraldehyde for 15 min. The fixative was removed, and cells were washed with PBS and stained with 0.1% crystal violet in PBS for 30 min. Plates were left to air dry overnight and incubated with 50 μl 1% Triton X-100 in PBS on a plate shaker for 30 min. Absorbance was read at 595 nm on a GloMax microplate reader (Promega).

Fully differentiated cells HB2-C, -L3, L3bp, and -L3-L3bp were seeded into collagen I coated (KOKEN, Tokyo, Japan) XFe24 Microplates (Seahorse Bioscience, North Billerica, MA, USA) at a density of 50,000 cells/well and were incubated in a CO2 incubator for 24 h, and then cells were washed three times and incubated in Seahorse XF DMEM Medium, pH 7.4 (Seahorse Bioscience) supplemented with 25 mM glucose and 1 mM sodium pyruvate for 1 h. The OCR was measured using a Seahorse XFe24 flux analyzer (Seahorse Bioscience). The compounds used to determine OCR included 1 μM isoproterenol (Sigma), 5 μM oligomycin (Sigma), 10 μM FCCP, 0.5 μM antimycin A (Sigma), and 0.5 μM rotenone (Sigma). OCRs were normalized to the total protein content.