Cd11b fitc m1 70

Quantification of circulating blood leukocytes was performed by flow cytometry as described (Kipari et al., 2013 (link)). Briefly, 30 µl of trunk blood was added to 30 µl of sodium citrate (3.9% w/v). Red blood cells were lysed by addition of 0.5 ml of FACS^TM lysis buffer (BD Biosciences, Oxford, UK), according to the manufacturer’s protocol. Fluorescent conjugated antibodies (100 ng each, in a total of 50 µl PBS) were added and incubated on ice for 25 min. The antibodies used were: CD45-PE (30-F11; Biolegend, UK), CD11b-FITC (M1/70; Biolegend, UK), Ly-6C-APC (AL-21; BD Biosciences, UK), Ly-6G-PB (1A8; Biolegend, UK). Cells were collected by centrifugation, resuspended in 100 µl of neutral buffered 10% formalin (Sigma-Aldrich, Dorset, UK) and analysed using a BD LSR Fortessa cell analyser (BD Bioscience, Oxford, UK) with manual compensation for each antibody. Supplementary Fig. 3 shows the gating strategy used to identify different classes of leukocytes. Data analysis was performed using FlowJo software v8.2 (TreeStar, USA). To determine the absolute numbers of cells, 5 × 10⁴ fluorescent flow-check fluorospheres (Beckman and Coulter, UK) were added to each sample prior to analysis. Forward and side scatter were used to distinguish cell and fluorospheres populations and cell numbers were calculated from the ratio of cells to fluorospheres in each sample.

Flow cytometry analyses were carried out using a three-laser configuration Thermo Fisher Scientific Attune Nxt flow cytometry system. Data were analyzed using the FlowJo software (TreeStar). The gates were set using the fluorescence minus one (FMO) control strategy. FMO controls are samples that include all conjugated Abs present in the test samples except one. The channel in which the conjugated Ab is missing is the one for which the FMO provides a gating control. The following mAbs were used: CD3 PerCP-Cy5.5 (145-2C11, eBioscience, San Diego, CA, USA); CD4 APC-eFluor 789 (GK1.5, eBioscience, San Diego, CA, USA); CD8 Super Bright 702 (53-6.7, Invitrogen, Carlsbad, CA, USA); CD11b FITC (M1/70, BioLegend); Gr1 BV510 (RB6-8C5, BioLegend); CD11c Pe-Cyanine7 (N418, eBioscience, San Diego, CA, USA); CD206 PE (MR6F3, Invitrogen, Carlsbad, CA, USA); FoxP3 APC (FJK-16s, eBioscience, San Diego, CA, USA) and IL-10 Alexa Fluor 700 (JES5-16E3, eBioscience, San Diego, CA, USA).

Mice were perfused as described in section “Immunochemistry.” Brains were removed, hippocampi were dissected, and hippocampal tissue was homogenized using a Dounce homogenizer. Myelin was removed by magnetic separation using myelin depletion beads and LS columns (Miltenyi Biotec) according to the manufacturer’s protocol or through a continuous 40% percoll gradient adapted from Hammond et al. (2019) (link). In our preliminary studies, we found that both of these methods were equally effective in terms of yield and viability (data not shown). Following myelin removal, cells were washed with FACS buffer (1× PBS containing 0.05% BSA), incubated in Fc block (2.4G2, 1:100, BioLegend), and stained with CD11b-FITC (M1/70, Biolegend), CD45-APC/Cy7 (30F11, Biolegend) and P2Ry12-APC (S16007D, Biolegend). DAPI (BD) was used as our viability marker. Microglia were defined as DAPI^–CD45^intCD11b⁺ cells and were sorted on a FACSAria (BD) in the University of Rochester Medical Center Flow Cytometry Core facility.