Ib 80827

The over-expression construct PLCγ2^P522R point mutation was inserted into the human PLCγ2^WT plasmid (RC200442, Origene) using Q5® site-directed mutagenesis kit (E0554S, NEB) according to manufacturer’s protocol. The mutagenesis primers used are listed in supplementary Table 1. For over-expression experiments, in brief BV2 cells were seeded onto either a 12-well plate or 8-well chamber slide (IB-80827, iBIDI) at a density of 140,000 and 21,000 cells per well, respectively, and allowed to settle for 24 h. 2 h prior to transfection a complete media change was done with fresh DMEM without pen/strep. For transfection, 50 ηg/100 μL of cDNAs (Control, PLCG2^WT and PLCG2^P522R plasmid) were mixed with 25 μL/100 μL Opti-MEM^® and 0.27 μL/100 μL of Lipofectamine™ 2000 (ThermoFisher) and incubated for 30 min. Afterwards, the media on the cells were replaced with transfection mix and incubated for 24 h. The media was then exchanged for fresh DMEM, and cells were incubated for a further 24 h before downstream assays, including uptake and metabolic profiling.

For the phagocytosis assay 5 × 10⁴ iMG progenitors were seeded onto 8-well chamber slide (IB-80827, iBIDI) coated with PDL and matured for 7 days with 50% medium change every other day. Uptake assays were then performed by exchanging 100% media to fresh media supplemented with either 250 ηM Aβ_1–42 HyLite Fluor 647 (AS-60493, AnaSpec), mouse brain purified ^tdTomato-tagged synaptosomes generated in house, FITC-Dextran 4kD (46944, Sigma), FITC-Dextran 150 kD (46946, Sigma), Zymosan beads-568 (Z23374, ThermoFisher) and 75 nM LysoTracker DND-99 (L7528, ThermoFisher) for 100 min. After elapsed time, iMG were washed with PBS and fixed with 4% paraformaldehyde for 30 min. Fixed iMG were subsequently stained with SNL (FL-1301-2, Vector) to visualise the membrane and with 2 µM Hoechst (62249, ThermoFisher) nuclear stain. Images were acquired using Nikon A1R inverted confocal microscope (Nikon) running the NIS elements software using 60 × oil immersion lens. Acquired images were analysed using FIJI and IMARIS, thresholding to WT/Control, marking ROI and quantified either as % Cell Area or uptake (cargo) volume per total cell volume. Statistical significance was assessed using a Kruskal–Wallis test.