Synaptosomes

Three to five 8-week-old animals were pooled per experiment. In brief, mice were intracardiac perfused with 10 ml ice-cold PBS. The hippocampi and cortices were dissected on ice. Tissue was weighed and homogenized in five volumes of sucrose homogenization buffer (5 mM HEPES pH 7.4, 320 mM sucrose and 1 mM EDTA) using a Dounce homogenizer with 15–20 strokes. The homogenate was centrifuged at 3,000g for 10 min at 4 °C and the supernatant was saved as total homogenate fraction (THF). The THF was centrifuged again at 14,000g for 12 min at 4 °C and supernatant was saved as cytosolic fraction. The pellet was carefully resuspended in 550 μl of Krebs–Ringer buffer (KRB: 10 mM HEPES, pH 7.4, 140 mM NaCl, 5 mM KCl, 5 mM glucose and 1 mM EDTA) and 450 µl of Percoll solution (for a final concentration of 45%). The solution was mixed by gently inverting the tube and an interface was slowly created with 400 µl of KRB. After centrifugation at 14,000g for 2 min at 4 °C, the synaptosomal fraction was recovered at the surface of the flotation gradient and carefully resuspended in 1 ml of KRB to wash. The functional synaptosomal preparation was centrifuged at 14,000g for 1 min at 4 °C. Fresh mouse synaptosomes were immediately divided into Eppendorfs at 2–2.5 mg of protein and resuspended in total 1 ml KRB in 50 nM of oAβ 40-S26C dimer or PBS as control and left overnight at 4 °C on nutator. Synaptosomes were then centrifuged at 14,000g for 1 min at 4 °C, supernatant was discarded and synaptosomes were washed in 1 ml PBS, after which they were centrifuged at 14,000g for 1 min at 4 °C to obtain oAβ-synaptosomes and control-synaptosomes. Briefly, 1 mg of synaptosomes were left at room temperature on nutator for 2 h in sodium bicarbonate 0.1 M with pHrodo Red, succinimidyl ester at a concentration of 1 mg ml⁻¹. After conjugation, synaptosomes were centrifuged at 14,000g for 1 min and resuspended for use in the in vitro synaptosome engulfment assay.

Primary mouse microglia were treated with 1 µg of pHrodo-conjugated S26C oAβ-treated synaptosomes. Plates were then placed in a CD7 with the incubator at 37 °C and 5% CO₂. Fluorescent (594 nm and 647 nm) and brightfield (oblique and phase) images were acquired at a ×20 objective (×0.5) at intervals of 3–5 min. A three-slice z stack was taken at a 1.5-μm interval to ensure that imaging was within focus throughout the imaging session; however, one plane was used for analysis. For analysis, the z-profile axis was plotted for respective pHrodos on ImageJ with respect to time. Fluorescence intensity at t = 0 was subtracted from subsequent time frames.