Ly6c apc clone hk1

Microglia were gated as CD45^intCD11b^high. In order to prove that the resulting cell fraction was not contaminated by other CNS-resident myeloid populations, we performed a flow cytometry staining of lymphocyte antigen 6 (Ly6). The panel comprised the following fluorochrome-conjugated monoclonal antibodies for the detection of cell-type-specific surface markers: Ly6G BV421 (clone 1A8, 1:100, BioLegend), CD11b FITC, Ly6C APC (clone HK1.4, 1:200, BioLegend), and CD45 BV510. One microgram of anti-CD16/32 antibody was added per 1 × 10⁶ cells for blocking of Fc receptors. Live/dead cell discrimination was performed with the help of eBioscience Fixable Viability Dye eFluor 780. All antibody concentrations were carefully titrated prior to experiments. Cells were stained for 15 min in the dark at RT and washed once with 500 µL PBS followed by centrifugation.
Finally, stained cells were resuspended in 300 µL 1X PBS and analyzed by a CytoFLEX S using Kaluza software. Flow cytometry compensations were set beforehand with eBioscience OneComp eBeads and the ArC Amine Reactive Compensation Bead Kit. Corresponding single stainings, FMO controls as well as unstained samples were used for further compensations and data interpretation. Neutrophils (gated as CD45^highCD11b⁺) served as positive control.

Microglia were isolated as previously described [54 ]. After perfusion, brains were removed from the skull and kept in cold medium A (HBSS (Gibco, 14170-088) with 0.6% glucose (Sigma, G8769) and 7.5 mM HEPES (Lonza, BE17-737E)). All subsequent steps were performed on ice, centrifugation was at 4 °C. Brains were dissociated using a Potter–Elvehjem tissue homogenizer after which the homogenate was passed over a 70 µM cell strainer (Corning, 352350) and pelleted by centrifugation at 220×g for 10 min. Next, myelin was removed by resuspending the pellet in 25 mL 24% Percoll (Fisher, 17-0891-01) in medium A (1 × final concentration) with 3 mL PBS layered on top, followed by centrifugation for 20 min at 950×g (acceleration 4 and brake 0). The microglia enriched cell pellets were incubated with CD11b-PE (clone M1/70, eBiosciences, 12-0112-82), CD45-FITC (clone 30-F11, eBiosciences, 11-0451-82), and Ly6c-APC (clone HK1.4, Biolegend, 128016) antibodies for 30 min on ice. Then the cells were washed once in medium A without phenol red and filtered into FACS tubes. Microglia were sorted by gating the DAPI^negCD11b^highCD45^intLy6c^neg cells using the Beckman Coulter MoFlo Astrios or XDP. Microglia were collected in siliconized Eppendorf tubes (Sigma, T3406-250EA) containing medium A. Flow cytometry data were analyzed using FlowJo Analysis Software.