Phagocytosis of pHrodo E. coli, synaptosomes and pHrodo™-Green-labelled fAβ

Synaptosomes were isolated from WT mouse brain tissue or human brain (obtained from Victorian Brain Bank), according to the Syn-PER Synaptic Protein Extraction Reagent (Thermo Fisher Scientific, #87793) protocol. The protein concentration was measured by nanodrop, and synaptosomes were labelled with pHrodo™ Red succinimidyl ester (Thermo Fisher Scientific, #{"type":"entrez-protein","attrs":{"text":"P36600","term_id":"12644234","term_text":"P36600"}}P36600) as described in¹⁴⁴ or with blue fluorescent 2.0 µm FluoSpheres™ Carboxylate-Modified Microspheres (Life Technologies, F8824) according to manufacturer instructions. Mouse pHrodo-conjugated synaptosomes were resuspended at 3.5 μg/μl in 5% DMSO in PBS and stored at −80 °C until use and human pHrodo-conjugated synaptosomes were resuspended at 5.5 μg/μl in 1% (w/v) BSA and stored at 4 °C until use. To examine the phagocytic properties of the XO4⁺ and XO4⁻ microglia populations, the microglia-enriched cell suspensions were isolated from methoxy-XO4-injected mice as described above, stained for CD45 and/or CD11b and seeded in 96-well plates. Following 30 min of resting at 37 °C and 5% CO₂, microglia were incubated with pHrodo-conjugated synaptosomes (4.25 μg per well), pHrodo™ Green E. coli BioParticles™ Conjugate (Thermo Fisher Scientific, #{"type":"entrez-protein","attrs":{"text":"P35361","term_id":"548429","term_text":"P35361"}}P35361, 66.7 ng per well) or pHrodo^TM-Green-labelled fAβ. Cells were collected after 45 min incubation at 37 °C and 5% CO₂ and stained with antibodies to microglia cell surface markers (CD11b-PE-Cy7, 1:200, Biolegend, #101216; CD45-APC-Cy7, 1:200, Tonbo Biosciences, #25-0459-T100). XO4⁺ and XO4⁻ microglia uptake of pHrodo-conjugated synaptosomes, pHrodo™ Green E. coli BioParticles™ Conjugate, or pHrodo^TM-Green-labelled fAβ was analysed using the BD™ LSR II analyser. iMGLs, BV2 cells or primary human microglia were incubated with conjugated synaptosomes (3.44 μg/μl) for 1.5 h, and uptake of synaptosomes was analysed using the BD™ LSR II analyser or during sorting on the BD FACSAria^TM Fusion or BD Influx^TM Cell Sorter.