Cd11b beads

Microglia were isolated from six organotypic brain slices, pooled from 3 individual wells or from freshly isolated mouse brain tissue using the Miltenyi OCTOMacs system. Tissue was added to C-tubes containing buffers and enzymes according to Miltenyi specifications. After homogenization, the cell suspension was passed through a 30-micron cell strainer into prechilled, sterile tubes. Myelin was removed using myelin removal beads (Miltenyi) and LS columns on a MACS separator. All wash buffers were prechilled and used according to manufacturer recommendations. Microglia were isolated using Miltenyi CD11b + beads and MS columns. Microglia were eluted from the column, spun, and resuspended in appropriate buffer for subsequent experiments.

Microglia were isolated from 1 striatum/sample via magnetic-activated cell sorting (MACS) using mouse anti-CD11b (for microglia) paramagnetic nanobeads (Miltenyi) according to the manufacturer’s instructions with some modifications. The MACS buffer used consisted of 1.5% bovine serum albumin (BSA) diluted in PBS from a commercial 7.5% cell-culture grade BSA stock (Thermo Fisher Scientific). For the microglial isolation, total striatal cell pellets after percoll (see above) were re-suspended in 90 μL MACS buffer and 10 CD11b beads (Miltenyi). Cells were then incubated for 15 min at 4 °C. Excess beads were washed with 1 mL MACS buffer and the cells pelleted at 300 rcf (relative centrifugal force) for 5 min at 4 °C. The cells were then passed through an MS MACS column attached to a magnet whereby CD11b positive cells stay attached to the column, whereas unlabeled cells flowed through the column. After washing the columns three times with MACS buffer, microglia were flushed from the column with 1 mL MACS buffer and pelleted at 300 rcf for 5 min at 4 °C. Cell pellets were lysed in QIAzol (Qiagen product code 79306), snap-frozen in dry ice and stored at -80 °C until RNA extraction.

RNA was extracted using PureLink Plus columns and converted to cDNA using the High Capacity RNA-to-cDNA kit (Life Technologies). qPCR analysis was undertaken using SYBR-green PerfeCTa (Quanta) and TLDAs were used as per manufacturer’s instructions on a 7900HT Real time machine (Applied Biosystems). ELISAs were undertaken using Duoset kits (R&D Systems). For flow cytometry, skin was enzymatically digested to release cells and stained using a subset of antibodies. Cells were stained with Fixable Viability Dye eFluor780 (eBioscience), fixed in 4% methanol-free paraformaldehyde (Thermo Scientific) or Cytofix/Cytoperm (BD), and analyzed on a MACSQuant Analyzer 10 (Miltenyi). For cell sorting, cells were labeled with CD11b beads and sorted on magnetized columns (Miltenyi). For immunohistochemistry, skin was fixed before freezing in embedding medium and sectioned. Sections were blocked in Tris-Saline-Tween (TBS)/5% fish gelatin (Sigma-Aldrich) incubated with a primary antibody against Lyve-1 and the secondary chicken anti-goat IgG Alexa Fluor 647-conjugated antibody (Life Technologies).