Ex cell 325 pf cho serum free medium

As for the human liver secretion studies, a piece of liver from the same liver lobe of Control-AAV and ARSA-AAV mice was embedded in 3% agarose (SeaPlaque™ Agarose, Lonza Bioscience) and 300-μm-thick liver slices were generated using a Krumdieck Tissue Slicer (TSE Systems China Ltd). Liver slices were maintained in oxygenated M199 media (Thermo Fisher Scientific, Scoresby, Australia) for 60 min before collection of secreted factors in oxygenated protein-free EX-CELL^® 325 PF CHO Serum-Free medium (Sigma-Aldrich, Castle Hill, Australia) for 16 h. This conditioned medium (CM) was utilized for lipidomics assessment, as well as applied to C2C12 myotubes for subsequent analysis of myotube glucose uptake.

Primary murine hepatocytes were isolated by collagenase perfusion^{63 (link)}. Briefly, the liver was perfused through the inferior vena cava with EGTA buffer (HBSS buffer + 0.5 mmol/l EGTA) for 15 min, followed by collagenase digestion (50 mg Collagenase H, Roche) in calcium buffer (HBSS buffer + 2 mmol/l CaCl₂) for 9 min. Hepatocytes were plated on collagenase-coated six-well plates with 500,000 cells per well, firstly in adherence media (Gibco M199 media, 100 U penicillin/streptomycin, 0.1% BSA, 2% FBS, 100 nmol/l Dexamethasone, 100 nmol/l Insulin) for 4 h, then in EX-CELL^® 325 PF CHO Serum-Free medium (Sigma-Aldrich, Castle Hill, Australia) for 16 h. The purity of the hepatocyte isolation was assessed as described previously^{8 (link)}. Conditioned medium containing all secreted factors was spun at 2000 × g for 10 min and the supernatant frozen for subsequent mass spectrometry proteomics assessment and cell culture conditioned media experiments. Hepatocytes were washed in PBS and frozen for mass spectrometric assessment of the intracellular proteome.