Phagocytic assay

Ex vivo assays: the phagocytic capacity of microglia was determined by the level of pHrodo™ Red fluorescence accumulated in the cells. First, we used pHrodo-conjugated E.Coli bioparticles. A suspension of 2.5 × 10⁵ sorted microglia cells in 100 μL of RPMI/1% BSA was incubated with bioparticles in an incubator at 37 °C and 5% of CO₂ for each time point. Cells were then pelleted by centrifugation at 1000×g for 10 min at 4 °C, resuspended in 200 μL of RPMI/1 %BSA and stained with CD11b-APC [clone M1/70, eBioscience] and rat CD45-PerCP-Cy™5.5 [clone 30-F11, BDBiosciences] antibodies for quantification by flow cytometry. We also developed a model using pHrodo-conjugated synaptosomes as a substrate for the phagocytosis. Synaptosomes were prepared from P21 mice hippocampus, and labeled with pHrodo Red, succinimidyl ester (Thermo Fisher Scientific, {"type":"entrez-protein","attrs":{"text":"P36600","term_id":"12644234"}}P36600) according to manufacturer instructions. Synaptosomes were resuspended at a concentration of 10 mg/ml in DMEM. Microglia were plated at a density of 5.10^{^5} cells/well in 24-well plates and received 150 μg of synaptosomes for 120 min in an incubator at 37 °C and 5% CO_2. When needed, baicalein was applied at a final concentration of 20 μM in 0,2% DMSO 90 min prior to the beginning of the assay (i.e. the addition of synaptosomes). Cells were collected by gentle trypsin treatment, rinsed and resuspended in PBS/1%BSA buffer to be stained with CD11b-V450 [clone M1/70, BDBiosciences], CD45 PerCP Cy5.5 [Clone 30-F11, BDBiosciences], Ly6G-APC [clone 1A8, BDBiosciences] and Ly6C-APC-Cy™7 [clone AL-21, BDBiosciences] both for negative exclusion and analysis by cytometry. After selection of the CD11b+/CD45low, cells were gated on PE channel for quantification of pHrodo fluorescence.

Experiments in Figs. 3c and 6f have been performed on the same batches in order to spare animals. Hence, data used for Fig. 3c control groups are a randomly chosen subset of Fig. 6f’s data.

In vitro, two different substrates where used: FCS-coated Yelloworange fluorescent carboxylated microspheres (Fluoresbrite® YO Carboxylate Microspheres 3.00 µm, Polysciences Europe GmbH, #19393-5) or the pHrodo-conjugated synaptosomes as described above. To quantify their phagocytic index, primary microglial cell cultures were incubated with a suspension of microspheres at a concentration of 1.1 × 10⁷ microspheres/ml for 30 min at 37 °C, intensively washed, and finally fixed with 4% paraformaldehyde. Cells were stained with anti-Iba1 antibodies. A blinded experimenter counted beads per cell using NIS Elements AR 3.26 software. To measure phagocytic activity by cytometry, cells were incubated for 60 or 120 min with 150 µg of synaptosomes per well. Medium was removed and cell collected following trypsin treatment, pelleted, rinsed in PBS/1%BSA, and stained with CD11b-FITC [clone M1/70, BDBiosciences] antibody for cytometry analysis.

FACS data were acquired using an LSR Fortessa 2-Blue 6-Violet 3-Red 5-YelGr laser configuration (BD Biosciences). Diva 8.0 (BD Biosciences) and FlowJo 10.5 (FlowJo, LLC) were used for data analyses.