2.2. Purification and ZetaView Analysis of Extracellular Vesicles (EVs) from MonoMac-1 (M0) and M1 and M2 Cells

Culture media of undifferentiated MonoMac-1 (M0) or differentiated M1 and M2 cells were obtained from 1 × 10⁷ cells of each cell types. EVs were purified from these supernatants utilizing the differential centrifugation method. Approximately 10-mL growth media from each cell type was first centrifuged at 3000× g for 30 min at 4 °C (Eppendorf Centrifuge, 5804R) to clear cell debris followed by a centrifugation at 10,000× g for 30 min at 4 °C (HB-6 rotor, Sorval Centrifuge, RC6+, Thermo Scientific), followed by filtration (Corning Incorp., NY, USA). At this step, clear supernatants were either stored at 4 °C or proceeded for ultracentrifugation. Ultracentrifugation was performed at 100,000× g for 4 h in a Beckman Ultracentrifuge. After centrifugation, the tubes were inverted to remove the remaining liquid and washed with PBS. The EV pellets were resuspended in 200-ul PBS. Regarding the zeta view analysis, EVs were diluted (1:250) in PBS to a final volume of 2 mL. For each measurement, three cycles were performed by scanning 11 cell positions each and capturing 60 frames per position (video setting: high) after capture; the videos were analyzed by the in-build Zeta View Software 8.02.31 with specific analysis parameters: maximum particle size: 1000, minimum particle size 5, and minimum particle brightness: 20. Hardware: embedded laser: 40 mW at 488 nm and camera: CMOS.