S2 cell lines obtained from DGRC were cultured in M3 media (S8398; Sigma-Aldrich, USA) containing 10% insect medium supplement (I7267; Sigma-Aldrich) at 25°C. Transfection was performed with cellfectin (Invitrogen, USA) according to the manufacturer’s instruction. pUAST-sona-HA (Kim et al., 2016 (link)) and pUAST-ana3-GFP (Stevens et al., 2009 (link)) were used for transfection.
To prepare the P100 fraction, conditioned media (CM) were sequentially centrifuged at 300g, 2,000g, 10,000g to remove dead cells and cell debris (Gross et al., 2012 (link)). The cleared CM was centrifuged at 100,000g to obtain two fractions. The supernatant fraction (SNΔ) containing soluble proteins was concentrated with an Amicon ultra-4 centrifugal filter unit (Millipore, USA) for further analysis. The pellet P100 fraction containing extracellular vesicles including exosomes was washed with phosphate-buffered saline (PBS) and then pelleted again by one additional centrifugation at 100,000g to remove contaminants.
To prepare the P100 fraction, conditioned media (CM) were sequentially centrifuged at 300g, 2,000g, 10,000g to remove dead cells and cell debris (Gross et al., 2012 (link)). The cleared CM was centrifuged at 100,000g to obtain two fractions. The supernatant fraction (SNΔ) containing soluble proteins was concentrated with an Amicon ultra-4 centrifugal filter unit (Millipore, USA) for further analysis. The pellet P100 fraction containing extracellular vesicles including exosomes was washed with phosphate-buffered saline (PBS) and then pelleted again by one additional centrifugation at 100,000g to remove contaminants.