Organotypic hippocampal slice cultures

Organotypic hippocampal (brain) slice cultures (OSHCs) were adapted from published protocols¹³⁹. On day 0, brains from 6-month-old 5xFAD and WT animals were coronally sectioned through the hippocampus on a vibratome (Leica; settings: speed = 0.4 mm/s and amplitude = 1.00 mm) in ice-cold cutting medium (MEM 1x, Life Technologies) containing 10 mM Tris, 50 U/ml Penicillin and 50 µg/ml Streptomycin) to obtain 400 μm thick brain slices. Brain slices were cultured in vitro with culture medium (25% (v/v) MEM 2x, 25% (v/v) HBSS 1x (Life Technologies), 25% (v/v) Horse serum (Life Technologies) with 10 mM Tris, 25 U/ml Penicillin and 25 µg/ml Streptomycin and 0.455% (v/v) 7.5% NaHCO₃ aqueous solution) on Millicell Cell Culture Insert (Merck) at air–medium interface. The media was completely replaced every second day. After 3 days of resting, Aβ plaques on brain slices were stained using an alternative fluorescent amyloid plaque-labelling dye, NIAD-4 (10 μM, BioVision, #2710) for 3 h, prior to the addition of ex vivo microglia-enriched fraction isolated from 6-month-old 5xFAD and WT animals. Microglia-rich fractions enriched by Percoll gradient (described in Acute isolation of microglia and fluorescence-activated cell sorting) were stained with CFSE (final concentration 5 μM; Life Technologies) for 20 min at 37 °C, and 2 × 10⁴ cells were added per hippocampus onto NIAD-4-stained hippocampal slice cultures for 5 days. As a control, synaptosome-labelled pHrodo-red particles were added to OHSCs for 5 days. Endogenous and replenished microglia were purified from OSHCs by mechanical dissociation using 70 μm mesh and enriched for microglia by density gradient centrifugation in 30% (v/v) isotonic Percoll at 1000g for 15 min. Cell pellets were incubated with Fc block (1:200; BD Biosciences, #553141) for 15 min on ice prior to staining with CD11b-PE (1:50) and CD45-BV786 (1:200) for 15 min. Cells were washed once in PBS and resuspended in 400 μl Zombie IR dye (1:1000; Thermo Fisher Scientific) for live cell discrimination. Endogenous (CFSE⁻) and exogenous (CFSE⁺) microglia (single, live, CD11b⁺, CD45^lo) that were either positive or negative for NIAD4 were sorted into 96-well plates (10 cells/well) containing 10 μl of Lysis Buffer from the Single Cell to Ct kit (Thermo Fisher Scientific), using the FACSAria™ III cell sorter. As a control, pHrodo-red-containing microglia were also sorted from the slices. The sorting/gating strategy is shown in Supplementary Fig. ^9a. cDNA synthesis and pre-amplification (18 cycles) were performed in accordance with manufacturer’s instructions and pre-amplified cDNA was diluted 5-fold prior to qPCR. The primers and probes used are listed in Supplementary Data ¹². Three independent biological experiments were performed. If enough cells were present from any populations, additional technical replicates were sorted. We report all the data, including experimental (solid circles) and technical (open circles) replicates, when available, for these experiments in Fig. 3d–g. The percentage of sorted cells belonging to Cluster 1 or Cluster 2 is calculated based on all replicates.