Seahorse XF Glycolysis Stress Test (ECAR) for Glycolytic Flux Quantification in MDA-MB-231 Breast Cancer Cells

Seed MDA-MB-231 at 2 x 10^4 cells/well in an XF96 microplate, with 4 background-correction wells (medium only). Minimum 5-6 technical replicate wells per condition, interleaved across the plate; 3 independent biological replicates on separate days. Glycolytic medium contains NO glucose and NO bicarbonate at baseline. Time course: 3 baseline measurements (no substrate), then 3 measurements after each of glucose (10 mM final), oligomycin (1.5 µM final), and 2-DG (50 mM final). For combined energetics, an XF Glycolytic Rate Assay (with rotenone/AA) can substitute to compute proton efflux rate (PER) and correct for mitochondrial CO2 acidification.

BSL-1/2 — MDA-MB-231 is a human tumor line; use universal precautions and a biosafety cabinet. Oligomycin is a potent toxin (handle stock in a fume hood, nitrile gloves). 2-DG is a metabolic inhibitor; avoid ingestion/inhalation. DMSO promotes dermal absorption of co-solutes. Collect drug-containing medium as chemical hazardous waste and plates/cartridges as biohazard solid waste; do not pour toxin-containing medium down the drain.

Background wells (cell-free, medium only) correct for non-cellular acidification/drift. Vehicle control: DMSO-matched wells for any drug-treated condition. Built-in positive control: the 2-DG injection should suppress ECAR to non-glycolytic baseline in every well, validating the glycolytic origin of the signal. The glucose injection itself is a within-assay positive stimulus (ECAR should rise sharply). Include an untreated reference condition and, where relevant, a known glycolysis inhibitor (e.g., 2-DG pretreatment or LDHA inhibitor) as a biological positive control.

MDA-MB-231 baseline (no-glucose) ECAR is low; glucose injection raises ECAR sharply (glycolysis); oligomycin further increases ECAR to glycolytic capacity (forcing maximal glycolysis); 2-DG collapses ECAR to non-glycolytic levels. Typical glycolysis values are 30-80 mpH/min/well at 2 x 10^4 cells; glycolytic reserve is often 20-60% above basal glycolysis. CV across replicate wells should be <15%. A glycolysis-impairing treatment reduces glucose-stimulated ECAR and capacity.

• MDA-MB-231 cells (ATCC HTB-26), passage <20, mycoplasma-negative
• Seahorse XF96 microplate + XFe96 sensor cartridge (Agilent 102416-100)
• Seahorse XF DMEM medium, pH 7.4 (Agilent 103575-100), with 2 mM glutamine but NO glucose/pyruvate at baseline
• D-glucose (Sigma G8270), 1 M sterile stock; 2-deoxy-D-glucose (Sigma D8375), 500 mM stock
• Oligomycin A (Sigma 75351), 5 mM in DMSO
• Seahorse XF Calibrant (Agilent 100840-000)
• Growth medium: DMEM (Gibco 11965) + 10% FBS + 1% pen/strep
• Hoechst 33342 (Thermo 62249) or BCA kit (Pierce 23225) for normalization
• Non-CO2 37 °C incubator

To quantify glycolytic function in adherent MDA-MB-231 triple-negative breast cancer cells using the Agilent Seahorse XF Glycolysis Stress Test, in which sequential injection of glucose, oligomycin, and 2-deoxyglucose (2-DG) resolves glycolysis (post-glucose ECAR), glycolytic capacity (post-oligomycin ECAR), glycolytic reserve (capacity minus glycolysis), and non-glycolytic acidification (post-2-DG). ECAR (mpH/min) serves as a proxy for the lactate-driven proton efflux rate.

1. Day 0: Hydrate sensor cartridge with 200 µL Calibrant per utility-plate well, overnight at 37 °C in a non-CO2 incubator.
2. Day 0: Seed MDA-MB-231 at 2 x 10^4 cells/well in 80 µL growth medium; background wells medium only. Pre-incubate at RT 1 h, then 18-24 h at 37 °C/5% CO2.
3. Day 1: Prepare glycolytic assay medium (XF DMEM + 2 mM glutamine, no glucose, pH 7.35-7.4 at 37 °C).
4. Wash cells twice with assay medium to 180 µL final; equilibrate 45-60 min at 37 °C non-CO2.
5. Load 10x injection stocks: port A glucose (100 mM), port B oligomycin (15 µM), port C 2-DG (500 mM). Add 20/22/25 µL to ports A/B/C.
6. Calibrate cartridge; run Glycolysis Stress Test template: 3x baseline, then A/B/C each with 3 cycles (mix 3 min, measure 3 min).
7. After the run, stain nuclei with Hoechst (10 µg/mL) and image-count per well, or run BCA, for normalization.

Independent variables: the metabolic perturbation (treatment vs. vehicle) and the sequential injections (glucose, oligomycin, 2-DG). Dependent variables: ECAR (mpH/min) and derived glycolysis, glycolytic capacity, glycolytic reserve, and non-glycolytic acidification (or PER if the Rate Assay is used). Controlled variables: seeding density, passage, glucose-free baseline medium and pH, temperature (37 °C non-CO2), injection concentrations, measurement timing, and normalization method.

We hypothesize that MDA-MB-231 cells, exhibiting a Warburg phenotype, will show high basal glycolysis and that a glycolysis-targeting perturbation (e.g., GLUT1 or LDHA inhibition) will reduce glucose-stimulated ECAR and glycolytic capacity by >30% relative to vehicle. The 2-DG injection will collapse ECAR to non-glycolytic levels in all wells, confirming the measured acidification is glycolysis-derived.

Export ECAR from Wave software. Compute glycolysis = (max ECAR after glucose) - (last baseline ECAR before glucose); glycolytic capacity = (max ECAR after oligomycin) - (non-glycolytic ECAR after 2-DG); glycolytic reserve = capacity - glycolysis; non-glycolytic acidification = ECAR after 2-DG. Normalize to nuclei or protein per well. Exclude wells with abnormal cell counts or non-responsive glucose injections. Aggregate technical replicates to one value per biological replicate. If using the Rate Assay, convert ECAR to PER using the buffer factor and subtract mitochondrial acidification.

1. No ECAR rise after glucose: baseline medium contained glucose, or cells are glucose-saturated — confirm glucose-free assay medium and proper washes. 2. Oligomycin doesn't increase ECAR: cells already near-maximal glycolysis (low reserve) or oligomycin degraded — make fresh stock; note low reserve can be biological. 3. High baseline ECAR: residual glucose or improper pH — verify medium pH 7.35-7.4 at 37 °C. 4. Edge-well variability: interleave conditions and pre-equilibrate plate at RT before incubation. 5. 2-DG doesn't fully suppress ECAR: increase 2-DG to 50-100 mM; residual acidification reflects non-glycolytic sources (CO2).

Treat biological replicate (n=3 independent seedings) as the unit. For two conditions, paired two-tailed t-test per parameter; for >2, one-way ANOVA with Tukey HSD. Set alpha = 0.05; report mean ± SEM and exact p-values. A priori power (d=1.2, power 0.8) supports n=3-4 biological replicates. Avoid pseudoreplication by not treating technical wells as independent n.