SEC-based Isolation of Microglia-derived Extracellular Vesicles: A Technique to Isolate Extracellular Vesicles from Microglia Conditioned Culture Media

1. Primary Culture of Microglia/Macrophages

1. Primary culture of microglia
   1. Culture commercial rat primary microglia (2 x 10⁶ cells) (see the Table of Materials) in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% exosome-free serum, 100 U/mL of penicillin, 100 μg/mL streptomycin, and 9.0 g/L glucose at 37 °C and 5% CO₂.
   2. Collect the conditioned medium after a 48 h culture and proceed to the isolation of EVs.
2. Primary culture of macrophages
   1. Culture commercial rat primary macrophages (1 x 10⁶ cells) in the medium provided by the manufacturer (see the Table of Materials) at 37 °C and 5% CO₂, with exosome-free serum.
   2. Collect the conditioned medium after a 24 h culture and proceed to the isolation of EVs.

2. Isolation of EVs

1. Pre-isolation of EVs from conditioned medium
   1. Transfer the conditioned culture medium from microglia or macrophage cultures (steps 1.1.2 or 1.2.2) into a conical tube.
   2. Centrifuge at 1,200 x g for 10 min at room temperature (RT) to pellet the cells.
   3. Transfer the supernatant into a new conical tube. Centrifuge at 1,200 x g for 20 min at RT to eliminate apoptotic bodies.
   4. Transfer the supernatant into a 10.4 mL polycarbonate tube and transfer the tube into a 70.1 Ti rotor. Ultracentrifuge at 100,000 x g for 90 min at 4 °C to pellet the EVs.
   5. Discard the supernatant and resuspend the pellet containing EVs in 200 μL of 0.20 μm filtered phosphate buffer saline (PBS).
2. Isolation of EVs
   1. Preparation of the home-made size exclusion chromatography column (SEC)
      1. Empty a glass chromatography column (length: 26 cm; diameter: 0.6 cm) (see the Table of Materials), wash and sterilize it.
      2. Place a 60 μm filter at the bottom of the column.
      3. Stack the column with a cross-linked agarose gel filtration base matrix to create a stationary phase of a 0.6 cm diameter and a 20 cm height.
      4. Rinse the phase with 50 mL of 0.20 μm filtered PBS. Store at 4 °C to be used later if necessary.
   2. Place the resuspended EV pellet on top of the stationary phase of the SEC column.
   3. Collect 20 sequential fractions of 250 μL while continuing to add 0.20 μm filtered PBS on the top of the stationary phase to prevent drying of the column. Store the fractions at -20 °C if necessary.
      NOTE: A longer storage than one week can be performed at -80 °C to maintain the EV integrity for molecular analysis.