Neuronal cell apoptosis

Primary neuronal cells in 6-well plates were incubated with (1) conditioned media (CM) collected from microglial cultures treated with sham EVs or CLP EVs at 2.5 × 10⁹ particles/mL, (2) sham EVs or CLP EVs (2.5 × 10⁹ particles/mL) alone, and (3) bafilomycin A1 (10 nM, Sigma). Sixteen hours after the treatment, the neuronal cells were washed with chilled DPBS and lysed for western blot with NP-40 lysis buffer, containing 50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 0.25% Na-deoxycholate, and complete protease inhibitor cocktail (Roche). Protein concentration was quantified by a Pierce BCA quantification assay (Thermo Scientific), and 60 μg of protein lysate was fractionated by SDS-PAGE (4–20% TGX stain-free gel, Bio-Rad). Proteins were transferred to a PVDF membrane using a semi-dry transfer unit, Trans-blot Turbo (Bio-Rad), at the default setting for low molecular weight proteins. Membranes were blocked with 10% nonfat milk containing 0.1% Tween 20 in Tris-buffered saline and then blotted with anti-cleaved caspase 3 antibody (Cat. #: 9664, 1:1,000, Cell Signaling, USA) at 4 °C overnight, followed by incubation with a HRP-conjugated secondary antibody (1:1000, Cell Signaling). Membranes were developed with Forte Western HRP substrate (Millipore Sigma), and proteins were visualized using a ChemiDoc imager (Bio-Rad). The intensity of the images was analyzed using Fuji Image J.

Primary neuronal cells were seeded in chambered borosilicate cover glass (Lab-Tek, Thermo Scientific) and incubated with conditioned media from microglia treated with sham EVs or CLP EVs as described above, and bafilomycin A1 (10 nM) for 16 h. Annexin-V (Annexin-V-FITC Fluorescence microscopy kit, Cat #: 550911, BD Pharmingen) and Hoechst 33258 (2 µl/mL, Sigma) were stained at room temperature for 15 min while protected from light, followed by washing with cold-PBS and cold-assay buffer provided in the kit. The images were collected using fluorescence microscopy (Ti Eclipse, Nikon) and analyzed with NIS Element (Nikon).