Xf cell glycolysis stress test kit

Eight thousand WI-38 cells (passaged not more than 10 times after thawing) per well were seeded into a Seahorse XF96 plate (EMEM, 10% heat-inactivated FBS, 1% MEM non-essential amino acid solution, 1% GlutaMAX-I) and left for 1 h at room temperature before incubation at 37 °C, 5% CO₂ for 24 h. After visual inspection by light microscopy, cells were washed twice with starvation medium (EMEM) and kept in starvation medium at 37 °C, 5% CO₂ for 24 h. After starvation, cells were treated with fresh starvation medium with or without 5 ng/ mL TGF-β1 and compound or vehicle (DMSO). A symmetrical plate layout was used that minimizes edge effects and optimizes assay robustness (Additional file 1: Figure S2). The edge wells were excluded for data analysis. For analysis of glycolysis parameters, Seahorse assays were performed according to the manufacturer’s instructions of the Seahorse Bioscience XF cell glycolysis stress test kit (#103020–100). Assays were performed in the presence of compounds (vehicle DMSO) and after visual inspection of the cells by light microscopy. During the assay cells were treated with 10 mM Glucose, 1.25 μM Oligomycin and 50 mM 2-Deoxyglucose. Mixing time was 3 min and measure time 4 min over a period of 3 cycles each.

ECAR and OCR were measured as described previously [18 (link)]. Cultured MBs were plated on XF24 cell culture plates (Seahorse Bioscience). OCR was measured simultaneously using a Seahorse XF24 Extracellular Flux Analyzer (Seahorse Bioscience), according to the manufacturer’s instructions. Mitochondrial function was assessed using an XF Cell Mito Stress Test Kit (Seahorse Bioscience), according to the manufacturer’s instructions. Oligomycin (6.25 μM), FCCP (1 μM), and rotenone/antimycin A (1 μM) were injected for a Mito Stress Test. The glycolytic function was assessed using an XF Cell Glycolysis Stress Test Kit (Seahorse Bioscience). Glucose (6.25 μM), oligomycin (1 μM), and 2-deoxyglucose (1 μM) were injected to test glycolysis. After analysis, the protein was extracted from MBs in each well using RIPA buffer (Thermo Fisher Scientific). The protein content was measured using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific).