Apc cy7 conjugated anti ly6c

Central nervous system tissues including brain and spinal cord were collected and macerated with cold PBS in small petri dishes that were kept cold (on ice). Cell suspensions were strained with a 70-µm strainer and mononuclear cells are isolated with 30% percoll gradient. The pelleted cells were washed and pre-blocked for Fc receptors using anti-CD16/32 (Fc-block). Cells were then incubated with monoclonal antibody at 4°C for 30 min, after which cells were washed with PBS containing 2% FBS. For flow cytometry antibodies the CD16/32 Fc-block, PE conjugated anti-F4/80, Pacific Blue conjugated anti-CD45, APC-Cy7 conjugated anti-Ly6C and/or Alexa 488 conjugated anti-CD206 were all obtained from Biolegend (San Diego, CA, USA). Anti-mouse Ron was purchased from R&D Systems (Minneapolis, MN, USA), and the secondary antibody was Alexa 488 conjugated to anti-goat for Ron. Stained cells were analyzed on a BD LSR Fortessa (BD Biosciences), and the flow output was analyzed with FlowJo software (Tree Star).

Microglia were depleted using PLX5622 (Plexxikon), an orally available CSF1R inhibitor. PLX5622 was formulated into AIN-76A rodent chow (Research Diets) at 1200 mg/kg and provided ad libitum during periods of depletion. For quantification of retinal microglia, freshly dissected retinas were dissociated using cysteine-activated papain as previously described (9 (link)). Cells were subsequently blocked with 1:100 anti-CD16/32 (BD Pharmingen, 553142) for 5 minutes on ice, followed by incubation with 1:200 PE-Cy5–conjugated anti-CD11b (BioLegend, 101209), 1:200 APC-Cy7–conjugated anti-Ly6C (BioLegend, 128025), and 1:200 APC-Cy7–conjugated anti-Ly6G (BioLegend, 127623) for 20 minutes on ice. After washes, samples were passed through a 40 μm filter and stained with 0.5 μg/mL DAPI in FACS buffer. Data were collected on a BD FACSAria II and analyzed using FlowJo 10.

Femurs of wild-type mice were collected and incubated for 30 min at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM) containing 1% collagenase D (from Clostridium histolyticum, Sigma-Aldrich, catalogue no. C5138-1G), 1 U ml⁻¹ of dispase (Thermo Fisher Scientific, catalogue no. 17105-041) and 1 U ml⁻¹ of DNase (Invitrogen) before cells were dissociated by trituration. Cells were dissociated to generate a single-cell suspension by filtering through a 40-μm nylon mesh. RBCs were eliminated using RBC Lysis Buffer (pluriSelect, Deutscher, catalogue no. 60-00050-11). Cells were subjected to Fc blocking (BioLegend, catalogue no. 101320, 1:200) and stained with FITC-conjugated anti-CD31 (BioLegend, catalogue no. 102506, 1:200), allophycocyanin (APC)-conjugated anti-CD45 (BioLegend, catalogue no. 103112, 1:200), phycoerythrin (PE)-conjugated anti-Ly6a (BioLegend, catalogue no. 122507, 1:200) and APC/Cy7-conjugated anti-Ly6c (BioLegend, catalogue no. 128025, 1:200) antibodies for 40 min. Analysis was performed with an SH800S Cell Sorter (SONY) and FlowJo software (Tree Star).