Xf cell culture plate

Cellular oxygen consumption rates were measured using a Seahorse XFe96 analyzer. The following protocol was adapted from the manufacturers protocol. Cells were seeded at 70,000 cells per well in a 96-well Seahorse XF cell culture plate. Cells were left at room temperature to evenly distribute the cells according to Lundholt et al., 2003 (45 (link)). The following day, medium was exchanged for carbonate-free DMEM (Merck, cat# D5030) containing 5 mM glucose, 4 mM glutamine. Cells were left for 60 min to equilibrate in a CO2 free incubator at 37 °C, before placing in the analyzer. To measure the dose–dependent decrease of respiration, a range of concentrations of the inhibitors were injected, and the remaining respiration measured. Finally, rotenone (50 μM) and antimycin A (50 μM) were injected to measure nonmitochondrial respiration. After completion of the measurement, the supernatant was aspirated, and 25 μl of lysis buffer (1% SDS, 0.1 N NaOH) was added. The plate was briefly vortexed, and the protein content was measured with a BCA protein assay (Thermo Fischer Scientific, cat# 23225). The respiration measurements were normalized to protein content per well, and values are reported in residual respiration after compound injection, relative to baseline in each well.

Extracellular acidification rate (ECAR) was assessed using a Seahorse XFe96 analyzer following manufacturer's protocols. PANC-1 cells were seeded in a Seahorse XF cell culture plate at a density of 1 × 10⁴ cells / well in normal growth media and left overnight to adhere. Once adhered cells were treated for 48 h with 0.25–25 μM pramlintide, davalintide, or rodent IAPP (rIAPP) in complete growth media or media with reduced FBS (10%–0.1% FBS) and glucose (25 mM–2.5 mM glucose). After 48 h, cells were washed and incubated in Seahorse XF base medium (Agilent 102353-100) supplemented with GlutaMAX (2 mM final concentration) for 1 h in a non-CO₂ incubator at 37 °C. ECAR was measured in Seahorse XF base medium with 10 mM glucose (MilliporeSigma, G7021), 1 μM oligomycin (MilliporeSigma, O4876), and 50 mM 2-deoxy-glucose (MilliporeSigma, D8375). H1299 and MIA PaCa-2 cells were assayed similarly, except for seeding density (5 × 10³ cells / well) and addition of 2 μM oligomycin was used during the assay. Glycolysis was measured by (maximum ECAR rate during glucose phase) – (last basal ECAR rate measured before glucose injection).

Briefly, cells were seeded in XF cell culture plates (Agilent Technologies, Inc.) at a density of 3x10⁴ cells/well and cultured at 37°C overnight. When cell monolayers were ~90% confluent, the culture medium was replaced with bicarbonate-free low-buffered assay medium (XF Base Medium; Agilent Technologies, Inc.) supplemented with 10 mM glucose, 2 mM glutamine and 1 mM pyruvate. Rotenone (cat. no. R105076; Shanghai Aladdin Bio-Chem Technology Co., Ltd.) at a final concentration of 0.5 μM and bufalin at final concentrations of 10, 20 and 40 μM were loaded to reagent ports on the sensor cartridge and added during oxygen consumption rate (OCR) measurement. Cells were incubated in a non-CO₂ incubator at 37°C for 1 h prior to testing. The OCR was detected by a Seahorse XF96 Analyzer (Agilent Technologies, Inc.) according to the manufacturer's instructions.