Xf24 extracellular flux analyzer kit

Branched polyethylenimine (PEI, M_w 1800 Da) and succinic anhydride (SA) were purchased from Innochem Technology Co., Ltd. (Beijing, China). 2,3-Dimethylmaleic anhydride (DMMA), PLGA (lactide : glycolide = 65 : 35, M_w 40000-75000 Da), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), cytochalasin D (Cyt. D), methyl-β-cyclodextrin (MBCD), sucrose, collagenase I, and collagenase IV were purchased from Sigma-Aldrich (Shanghai, Trading Co., Ltd). Polyethylene glycol monostearate (C₁₈-PEG₂₀₀₀-OH, CPO) was obtained from Tokyo Chemical Industry Co., Ltd. C₁₈-PEG₂₀₀₀-TPP (CPT) was synthesized as our previously reported 38 (link). Doxorubicin hydrochloride (DOX·HCl) was provided by Aladdin Reagent (Shanghai, China). N,N-dimethyl formamide (DMF), chloroform (CHCl₃), and isopropanol were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Lysotracker Green (Yeasen), Mitotracker Green (Beyotime), BCA protein assay kit (Beyotime), mitochondrial membrane potential assay kit with JC-1 (Beyotime), mitochondrial isolation kit (Beyotime), nuclei extraction kit (ExKine), XF24 extracellular flux analyzer kit (Seahorse Bioscience), and matrigel matrix (BD Biosciences) were all obtained from domestic suppliers. All other reagents and solvents were of analytical grade and used as received.

The Seahorse Bioscience XF24 Extracellular Flux Analyzer Kit (Seahorse Bioscience) was used to measure glucose dependency in vitro. The Mito Fuel Flex test was completed after subjecting cardiomyocytes to glucose oxidation pathway inhibition by UK5099 (4 μM) followed by long‐chain fatty acid oxidation inhibition by Etomoxir (2 μM). This order of inhibitors allows for calculation of glucose oxidation dependency. The protocol was modified to test dependency in Pdk4 inhibited cardiomyocytes. Prior to loading isolated cardiomyocytes to the Seahorse cell‐plate, cardiomyocytes were incubated with 10 μM Pdk4 inhibitor (MedChem Express, Cat# HY‐135954A) in DMEM medium (Seahorse Bioscience) for 5 min at 37°C. The cell‐plate was also loaded with cardiomyocytes suspended in 10 μM Pdk4 inhibitor in DMEM medium.