Glycolytic rate assay kit

Measurement of glycolysis was done using the Glycolytic rate assay kit (Seahorse, Agilent Technologies), following the manufacturer’s protocol. Briefly, cells were seeded in Xe96 plates treated with BDQ for 24 hr. The cells were then incubated in the assay medium (Seahorse XF Base Medium without phenol, 2 mM glutamine, 10 mM glucose, 1 mM pyruvate and 5.0 mM HEPES) at 37°C, during 1 hr. Extracellular acidification rate (ECAR, milli pH/min) and oxygen consumption rate (OCR, pmol/min) were measured using the Seahorse Bioscience XFe96 Analyzer.

Cells were seeded at a density of 4x10⁴ cells/well in gelatin-coated Seahorse XF24 polystyrene cell culture plates on the day before performing the assay. DMEM without NaHCO₃ or phenol red supplemented with 2 mM L-glutamine, 10 mM D-glucose, 1 mM sodium pyruvate and 5 mM HEPES buffer adjusted to pH 7.4 was used as assay medium. Cells were washed 3 times with this medium 1 hour prior to running the assay and incubated in a humidified non-CO₂ incubator at 37°C according to the manufacturer’s instructions. Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were measured in 5 cycles between each injection (2 minutes mixing, 2 minutes recovery and 2 minutes measurement), and the respective rates were determined. This was done using the company protocol for the glycolytic rate assay kit (Agilent) by injections of equimolar concentration of rotenone and antimycin A (Rot/AA) (5 μM) followed by injection of 2-deoxy-glucose (2DG) (50 mM). Measurements were normalized to cell density by fixing cells in 0.5% paraformaldehyde-lysine-periodate in PBS then adding 120 μl 0.1% crystal violet in PBS to each well incubated at room temperature for 3–4 minutes followed by washing with tap water. Crystal violet was solubilized by adding 33% acetic acid and OD was measured 550 nm using a microplate reader.

The XF96 Extracellular Flux Analyzer (Agilent, Santa Clara, CA) was used to determine the glycolytic profile of cells. Oxygen consumption rate (OCR), extracellular acidification rate (ECAR) and proton efflux rate (PER) were determined using the Glycolytic Rate Assay Kit (Agilent), according to the protocols provided by the manufacturer. Briefly, macrophages were seeded at a density of 1 x 10⁵ cells per well in the manufacture’s 96-well plate and allowed to attach overnight. Cells were then infected or not with B. abortus at a MOI of 100:1 for 24 hrs in normal cell culture medium. Prior to the assay, cells were washed tree times and media was changed to Seahorse XF DMEM medium pH 7.4 (Agilent), supplemented to contain 25 mM glucose, 4 mM L-glutamine and 2 mM pyruvate. The plate was allowed to equilibrate for 1 hr in a CO₂-free incubator at 37 °C before loading into the Seahorse analyzer. Seahorse XF96 cartridges were hydrated according to the manufacturer’s instructions. Three measurements of OCR and ECAR were performed before injection of mitochondrial inhibitors (rotenone and antimycin A) and then three additional measurements were achieved before injection of the inhibitor 2-DG. Experiments were performed with 4 replicates of each condition. The bioenergetic parameters mitoPER and glycoPER were calculated as described elsewhere [34 (link)].