Quantifying Receptor Recycling in Microglia

Receptor recycling assays were performed as previously described (3 (link), 53 (link)). Briefly, primary microglia were plated on PDL-coated coverslips in 24-well plates at a density of 20,000 cells per well. Cells were maintained in DMEM with 10% FBS and 1% Pen/Strep and supplemented with 10 ng/mL M-CSF for 24 hours. Cells were then incubated in DMEM with 10% donkey serum (S30-M, Sigma-Aldrich) for 15 minutes at 37°C. Antibodies against CD36 (ab23680, Abcam) or TREM2 (AF1729, R&D Systems) were added to the cells in DMEM with 1% donkey serum for 1 hour at 37°C. Cells were then acid washed with cold DMEM at pH 2.0. Cells were cultured in DMEM with 10% donkey serum for 1 hour at 37°C and then incubated with fluorophore-conjugated secondary antibodies (Alexa Fluor 555 or Alexa Fluor 647, Invitrogen) in 1% donkey serum for 1 hour at 37°C. Cells were again acid washed with cold DMEM at pH 2.0 and washed with cold PBS. Cells were then fixed with 4% paraformaldehyde (PFA), washed with PBS, and mounted on glass slides. Similar experiments were performed on BV-2 cells plated on glass coverslips and pretreated with vehicle or 10 μM C3aRA (SB290157, Calbiochem). Fluorescent signal from vesicles containing recycled receptors were thresholded and the intensity of the fluorescence signal was determined by ImageJ (NIH) and normalized to WT or vehicle-treated cells.