Xf real time atp rate assay report generator

Caco-2 cells were seeded into a Seahorse XF96 V3 PS Cell Culture Microplate and grown until confluent. The cells were either mock-treated or infected with SARS-CoV-2/FFM7 for 24 h before measurement at an MOI of 0.01. The day before measurement, a sensor cartridge was hydrated with Seahorse XF calibrant solution at 37 °C in a non-CO₂ incubator overnight. On the day of assay, the stock solutions of the Seahorse XF Real-Time ATP Rate Assay Kit (oligomycin, rotenone/antimycin A) were freshly prepared, diluted, and loaded into the corresponding sensor cartridge ports. Directly before measurement, the cells were washed with a warm Seahorse XF DMEM medium (pH 7.4, supplemented with 1 mM pyruvate, 2 mM glutamine, and 10 mM glucose) and subsequently incubated in this medium for 1 h at 37 °C in a non-CO₂ incubator. The assay was conducted using the Seahorse XFe96 Analyzer and the Real-Time ATP Rate Assay Kit protocol. The order of injections was: oligomycin and rotenone/antimycin A. The final concentrations in the assay were 1.5 µM for oligomycin and 0.5 µM each for the rotenone/antimycin A-mix. The data was evaluated via Seahorse Wave Software and normalized to cell number via DAPI count of each well. Calculation of mito- and glyco-ATP was done via the Seahorse XF Real-Time ATP Rate Assay Report Generator.

The ATP rate assay was performed using an XFe96 Extracellular Flux analyzer, in accordance with the manufacturer’s instructions (Seahorse Bioscience). MPI-KO HT1080 cells (#3, 1 × 10⁴ cells/96-well assay plate) were seeded and incubated for 18 hr in complete DMEM. Prior to the assay, the cells were preincubated for 60 min at 37°C in XF DMEM medium containing 10% FBS, 10 mM glucose, 4 mM L-glutamine, and 50 μM Man. The cells were then treated with 5 mM Man or medium alone for 120 min, followed by incubation with 1.5 μM oligomycin for 18 min and then 0.5 μM each of rotenone and antimycin A for 18 min. Data analysis was performed using the XF Real-Time ATP Rate Assay Report Generator (Seahorse Bioscience).