Agilent seahorse xf cell mito stress test

Day 20 ZZ, MZ and MM iHeps were seeded onto 96 well Matrigel coated Seahorse XF Cell Culture Microplate (Agilent Technologies) at a density of 50,000 cells/well. After 24h fresh media was applied for another 24h. The Agilent Seahorse XF Cell Mito Stress Test was then performed per manufacturer’s instructions. Briefly, iHeps were washed two times with prewarmed Seahorse Media (XF Base with 25mM Glucose and 10mM Pyruvate added), 180μL of assay media added, and cells were placed into a non-CO2 37 Celsius incubator for 60 min. The prepared sensor cartridge and cells were then loaded into the Agilent Seahorse XFe96 Extracellular Flux Analyzer for Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR) quantification. Port injections contained the following: Port A, oligomycin at a final concentration of 2μM; Port B, FCCP at a final concentration of 1μM; Port C, antimycin A and rotenone at a final concentration of 1μM. To normalize for cell number iHeps were fixed in paraformaldehyde for 10 min at room temperature then stained with Hoechst 3342. Widefield images of each well were then obtained using the Nikon IHC microscope and individual nuclei quantified using ImageJ.