Phagocytosis was assessed in rat primary microglial cells. For this, the cell culture media was first changed to serum-free DMEM supplemented with 1% N-2 supplement (Gibco). After 24 h, cells (3.5 × 105 cells/well) were incubated under control conditions or in medium containing 75 mM EtOH, TII, or TII with 75 mM EtOH. After 24 h, the medium was replaced with fresh medium (control or containing 75 mM EtOH) supplemented with 6-carboxyfluorescein (FAM)-labeled Aβ1–42 (0.5 μM; Anaspec, Fremont, CA, USA). The Aβ peptide was dissolved in DMSO to obtain a 0.1-mM stock, diluted into DMEM to a final concentration of 500 nM, then incubated at 37 °C for 1 h to promote aggregation. Cells were incubated for indicated times, followed by one washing with PBS to remove Aβ, then harvested using 0.5% Trypsin (Gibco). Blocking solution containing PBS and FBS (1:1 ratio) was applied for 10 min on ice. Cells were collected, resuspended in 400 μl ice cold FACS solution (PBS supplemented with 2% FCS), and measured by flow cytometry using the Gallios software (Beckman Coulter’s). Phagocytosis was analyzed and quantified for total uptake and for the percentage of cells with internalized Aβ, using Flowing Software (University of Turku, Finland).
