Clone ra3 6b2

For flow cytomentric analysis of CNS-invading cells, mice were killed with CO₂ and perfused intracardially with PBS. Spinal cord tissue was homogenized using MACS miltenyi C-Tubes and a gentleMACS dissociator according the manufacture’s guidelines and loaded on a 30:37:70% Percoll gradient. The 37:70% interphase was collected, the cells were washed and treated with Fc-block (1 μg per 10⁶ cells) before staining. Fluorescence staining was performed using the following antibodies purchased from BD or eBioscience: (CD11b, 1:1,000) eBioscience clone M1/70, (CD45.2, 1:200) eBioscience clone 104, (B220, 1:200) clone RA3-6B2, (CD4, 1:100) eBioscience clone GK1.5, (CD8, 1:300) BD clone 53-6.7. Analysis was performed on a FACSCantoII (BD Biosciences) and analysed by FlowJo software.

For functional analysis of pDCs (Ifn-α production and costimulatory molecule upregulation), bones (femur, tibia, collarbone) of Apoe^-/- control and Apoe^-/-BDCA2-DTR mice were harvested and flushed with Hank’s medium (Hanks’ Balanced Salt Solution + 0.3 mmol/l EDTA + 0.1% BSA). Cell suspensions were treated and stained according to FACS protocol with following antibody cocktail: anti-CD45 (eBioscience, clone 30-F11), anti-B220 (eBioscience, clone RA3-6B2) and anti-SiglecH (eBioscience, clone eBio440c). After cell sorting, sorted pDCs were cultured in 24 well flat-bottom plates (1x10⁵ cells/well) in RPMI1640 Medium with L-Glutamine (Gibco by life technologies) and 1% Penicillin/Streptavidin with or without 5 μg/ml CpG oligodeoxynucleotides (ODN 1585, InvivoGen). After incubating pDCs 12 hours at 37°C, cell supernatants were used for Ifn-α ELISA and costimulatory molecule upregulation was measured with FACS using anti-CD86 (eBioscience, clone GL1) and anti-MHC class II (BD Pharmingen, clone 2G9) antibodies. To analyze the expression of DTR on pDCs, splenic pDCs of Apoe^-/- control and Apoe^-/-BDCA2-DTR mice were stained and measured according to the FACS protocol with an anti-hDTR antibody (human HB-EGF, clone #125923, R&D Systems).

In each experiment, the proportions of splenocyte subpopulations (lymphoid and myeloid lineages) were evaluated by flow cytometry after 24 h of incubation (3 × 10⁶/3 mL/well) in CM at 37 °C and 5% of CO₂ in a six-well plate. Nonadherent cells were collected and counted, while adherent cells were first detached by incubation in warm Accutase solution at 37 °C for 15–20 min, followed by gentle scraping with a cell scrapper and washing with PBS. Cell samples (0.4 × 10⁶/sample) of naïve splenocytes and pooled nonadherent and adherent subpopulations after 24 h of incubation were stained with anti-mouse monoclonal antibodies to CD 45.2 (PE-Cyanine7, clone 104, eBioscience, San Diego, CA, USA), CD3 (PerCPeFluor710, clone 17A2, eBioscience, USA), CD45.R (B220, APC), and (clone RA3-6B2, eBioscience). The other samples were stained with anti-mouse antibodies against CD11c (PerCP Cyanine 5.5, clone N418, eBioscience, USA) and CD11b (FITC, clone M1/70, BioLegend, USA). Cells were stained with the antibodies for 30 min at room temperature and in the dark, washed and analyzed by flow cytometry. Phenotypic analysis was performed using a FACS Canto cell analyzer (Becton Dickinson Biosciences, Franklin Lakes, NJ, USA). The acquired data were analyzed using FACS Diva software.