Amyloid Beta Uptake by Glia Cells

    A. Prepare conditioned media and cells on Day 1

  1. Grow 293sw cells (using G418 DMEM) to complete confluence, then remove media and replace it with DMEM without any drug, 10 ml each plate.
  2. Add 2 ml of stock poly-Lysine solution into 200 ml of autoclaved H2O and sterilize solution with filter. Cover each petri dish with 5 ml of poly-Lysine for at least 20 min inside the hood.  Aspirate poly-Lysine and let it air dry inside the hood. 
  3. Transfer conditioned media of BV2 cells to a fresh 50ml conical tube, add 2 ml of trypsin to the plate and incubate for 2 min.  Use conditioned media to wash off cells and transfer to 50 ml tube. Centrifuge at 1000 rpm for 5 min. 
  4. Use 5 ml of media to wash poly-Lysine coated plates immediately before use.
  5. Aspirate media and use 10 ml new RPMI media to resuspend cell pellets and transfer to poly-Lysine coated plate.
  6. For other cell lines, directly split them onto new plates.

B. Day 2

  1. Collect conditioned media of 293sw cells into 50 ml conical tube inside hood. Centrifuge at 3000 rpm for 10 min.
  2. Transfer conditioned media of BV2 cells to a fresh plate if necessary; aspirate all conditioned media from BV2 or U373 cells.
  3. Transfer 8-10ml of 293sw conditioned media to BV2 or U373 cells.  Store 220 μl of conditioned media (duplicate wells and duplicate plates) for ELISA (on information sheet, ask for 1:64 dilution of amyloid beta total, no dilution for amyloid beta 42).
  4. Incubate for 24 hr, take 1.5 ml of conditioned media and spin at 14000 rpm for 5 min.  Transfer 220 μl conditioned media (duplicate wells and duplicate plates) for ELISA (on information sheet, ask for 1:64 dilution of amyloid beta total, no dilution for amyloid beta 42).
  5. Aspirate conditioned media and add 1 ml 20 mM EDTA in PBS.  Collect cells and spin at 6000 rpm for 5 min.  Store cell pellets at –80°C.