Xf dmem base medium

Oxygen consumption rates (OCRs) were measured by the Seahorse XF Analyzers (Agilent)^{32 (link),33} . Primary cultured adipocytes from gWAT of WT or TMEM86A AKO, differentiated C3H10T1/2 adipocytes overexpressing Mock or TMEM86A, and differentiated C3H10T1/2 adipocytes treated with either vehicle, LPC P-18:0 (10 μM), or LPE P-18:0 (10 μM) were washed and maintained in assay medium (XF DMEM Base Medium, pH 7.4 (Agilent, cat#103575-100)) supplemented with D-(+)-glucose (25 mM) and L-glutamine (4 mM) at 37 °C. XFp Cell Mito Stress Test Kit (Agilent, cat#103010-100) was used with the following concentrations: 2.5 μM of oligomycin, 0.5 μM of carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP), and 0.5 μM Rotenone/Antimycin A. Basal and maximal OCRs were calculated by subtraction of non-mitochondrial respiration. ATP production-related OCRs were calculated by subtracting the oligomycin A-induced OCR from the basal OCR. Spare respiratory capacity was calculated by subtracting Basal OCR from Maximal OCR. Proton leak was calculated by subtracting non-mitochondrial respiration from the oligomycin A-induced OCR. OCRs were normalized with protein concentrations. Agilent Wave software (version 2.6.0.31) was used to analyze the data.

XFp Analyzers were used to measure oxygen consumption rate (OCR). Cells were incubated in XF DMEM base medium (Agilent, 103575-100, pH 7.4, Cedar Creek, Texas, USA) supplemented with 4 mM L-glutamine (Sigma, G8540, St. Louis, MO, USA) and 25 mM D-glucose (Sigma, G7021, St. Louis, MO, USA) at 37 °C for the measurement. XFp Cell Mito Stress Test Kit (Agilent, 103010-100, Cedar Creek, Texas, USA) was sequentially prepared with the following optimal final concentrations: 2.5 μM oligomycin, 0.5 μM FCCP, and 0.5 μM rotenone/antimycin A, and basal, maximal, proton leak and were calculated previously described [14 (link)].

Urothelial cells were harvested from the mucosal surfaces of the inside-out mouse bladders. The cells were incubated in a Liberase solution at 37 °C for 15 min, after which the digestion was stopped by adding 2% fetal bovine serum and 0.2 μM EDTA. The cells were then cultured in cell culture microplates pre-coated with poly-l-lysine (Seahorse Bioscience, Santa Clara, CA, USA) at 3 × 10⁴ cells/well density in 500 µl XF Base DMEM medium (Agilent, Santa Clara, CA, USA) and incubated at 37 °C in 5% CO₂. The assays were conducted at different time points by adding glucose, Rot/AA, and 2-DG successively in an XFe24 extracellular flux analyzer (Agilent, Santa Clara, CA, USA), and the ECAR data were recorded automatically.