Macs ls

CX₃CR1⁺CD11c⁺ DCs were purified from spleens of CX₃CR1^+/gfp or CX₃CR1^gfp/gfp mice (50 (link)). Splenocytes were initially enriched for CD11c⁺ cells using a MACS LS column (Miltenyi Biotec, Bergisch Gladbach, Germany) after surface-staining with PE-conjugated anti-CD11c mAb according to the manufacturer's instructions. Enriched CD11c⁺ cells were then applied to a FACS sorter to purify CX₃CR1⁺CD11c⁺ DCs. Purified CX₃CR1⁺CD11c⁺ DCs contained CD11b⁺ cells as well. CX₃CR1^+/gfp and CX₃CR1^gfp/gfpCD11c⁺ DCs (5 × 10⁵ cells/mouse, 50 μl) were injected into left and right footpads of CX₃CR1^−/− mice, respectively. CX₃CR1^−/− recipients were immediately infected with JEV (5.0 × 10⁷ PFU) via footpad, and leukocytes were obtained from left and right popliteal LNs at 3 dpi. Popliteal LN cells were surface-stained with PE-conjugated anti-mouse CD11c for CX₃CR1^+/gfp and CX₃CR1^gfp/gfp CD11c⁺ DCs. In some challenge experiments, purified CD11c⁺ DCs (1.5 × 10⁶ cells, 250 μl) were injected i.v. into CX₃CR1^−/− mice. Flow cytometric analysis was performed using a FACS Calibur flow cytometer (Becton Dickson Medical Systems) with FlowJo software (Tree Star).

CD14⁺ monocytes were obtained from peripheral blood mononuclear cells (PBMCs) from healthy adult donors. PBMCs were obtained using a density gradient with Lymphoprep at a density of 1.077 + 0.001 g/mL (STEMCELL Technologies). CD14⁺ cells were isolated from PBMCs by negative magnetic selection using Micro Beads and MACS LS columns (Miltenyi Biotec, Bergisch Gladbach, North Rhine-Westphalia, Germany) following the protocol provided by the supplier. CD14⁺ cells were incubated in RPMI-1640 medium (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS) (Biowest, Nuaillé, PC, France), 2 mM L-glutamine (Biowest), 1× penicillin/streptomycin (Biowest) and 100 μg/mL gentamicin (Biowest) until use.

To isolate RGCs, we used a method that has previously been verified with slight modifications. Briefly, animals from each group were euthanized with CO₂ asphyxiation and their retinas were harvested in cold neurobasal medium and placed in a 37°C water bath for 5 minutes. The neurobasal media was removed and replaced with fresh pre-warmed neurobasal media containing papain (0.06mg/ml or 33.4 U/mg) and 5mM L-cysteine and incubated 20 minutes at 37°C. The papain solution was removed and replaced with neurobasal containing 2mM L-Glutamine (Gibco; Catalog no. 25030081) B-27 supplement (Gibco; Catalog no. 17504044) and 10% FBS (Gibco; Catalog no. 26140079). The retinas were dissociated by gently pipetting up and down with a wide bore 1ml pipette tip. Cells were then centrifuged at 450g for 8 minutes and resuspended in 90 μl of isolation buffer (DPBS + 0.5% BSA + 2mM EDTA) containing 25 μg/ml DNAse I + 5mM MgCl2. Cells were filtered through a 30μm cell strainer and incubated with CD90.2 magnetic beads (Miltenyi Biotec; Catalog no. 130-121-278) at 4°C for 10 minutes and then isolated using MACs LS (Miltenyi Biotec; Catalog no. 130-042-401) columns following the manufacturer’s instructions. After isolation, cells were washed with DPBS, centrifuged at 450g for 8 minutes and their pellets stored at −70°C prior to proteomics sample preparation and LC/MS analysis.