4.5. Exosome Purification and Analysis

Cells were seeded into 12-well plates at a density of 0.5 × 10⁵ cells/well. 1 μM of either Lacripep, A96, or LP-A96 were added during seeding. Cells seeded with dPBS or complete medium served as negative and positive controls, respectively. Cells were then incubated at 37 °C for 72 h, undisturbed. After this period, culture media were collected and subjected to exosome purification. After removal of culture media, cells were immediately collected in 50 μL radioimmunoprecipitation assay (RIPA) buffer supplemented with protease inhibitor cocktail (#78430, Thermo Fisher, Waltham, MA, USA) to measure total protein concentration.

To purify exosomes, collected culture media under each condition was cleared of cell debris and microparticles. To do this, the media was spun down at 300 g for 5 min. Collected supernatant after this centrifugation was then spun down at 2000× g for 10 min. Collected supernatant after this centrifugation was then spun down at 10,000× g for 30 min. Collected supernatants after these three centrifugations were concentrated and subjected to column purification (#qEVoriginal/70 nm, iZon Sciences, Medford, MA, USA) that is optimized for exosome purification. The purified exosomes were analyzed for the amount and the size using ZetaView^® nanoparticle tracking analyzer (PMX-120, Particle Metrix GmbH, Meerbusch, Germany). The number of exosomes was normalized to the protein concentration in cell lysates measured using the Micro BCA™ Protein Assay Kit (#23235, Thermo Fisher, Waltham, MA, USA). Exosomes in 0.1X dPBS were used for zeta potential analysis in ZetaView^®.

Exosomes were resolved by SDS-PAGE and blotted with antibodies to CD9 (1:250 dilution, #MA1-80307, Invitrogen, NY, USA), TSG101 (1:500 dilution, #ab3071, Abcam, Cambridge, MA, USA) and Alix (1:500 dilution, #2171, Cell Signaling Technology, Danvers, MA, USA). Primary antibodies were incubated overnight at 4 °C. Donkey anti-mouse (#925-68072, 1:5000 dilution) and goat anti-rabbit (#925-32211, 1:5000 dilution) secondary antibodies were purchased from LI-COR (Lincoln, NE, USA) for fluorescence imaging.

Exosomal RNAs were isolated using miRNeasy Serum/Plasma Kit (#217184, Qiagen, Hilden, Germany) and analyzed with 2100 Bioanalyzer system (Agilent Technologies, Santa Clara, CA, USA).