Hemavet 950fs whole blood counter

Six hours after the initial delivery of OVA and adjuvant, blood was sampled from the tail vein of treated mice, collected in EDTA-coated microcuvette tubes (Sarstedt), and analyzed in a Hemavet 950FS whole blood counter (Drew Scientific). For the analysis of IL-6 in plasma, blood was centrifuged at 14,000 × g for 10 min at 4 °C, after which the cytokine concentration was determined by ELISA (431302, BioLegend), according to the manufacturer’s instruction.

One day after the last treatment, blood was collected from the tail vein in EDTA-coated microvette tubes (Sarstedt), and analyzed in a Hemavet 950FS whole blood counter (Drew Scientific, Waterbury, USA). Spinal cord sections were dissected and stained with H&E, Luxol fast blue (LFB, for myelination assessment), and antibodies against amyloid precursor protein APP (evaluating axonal damage) or CD3, B220 or MAC3 for visualizing infiltrating T and B cells and macrophages, respectively^{18 (link)}. Flow cytometry was done on spleen cells 15 days after MOG inoculation (two hours after the 8th treatment). Doublets were excluded and living cells were selected based on live-dead stain (Invitrogen). pDC (CD3⁻ CD19⁻ B220⁺ SiglecH⁺), cDC1 (CD3⁻ CD19⁻ CD11b⁻ CD11c⁺ MHCII⁺ XCR1⁺) and cDC2 (CD3⁻ CD19⁻ CD11b⁺ CD11c⁺ MHCII⁺ XCR1⁻) percentages were determined, and the intracellular expression of designated cytokines determined. For Tregs, the CD3⁺ CD4⁺ CD8⁻ CD25⁺ FoxP3⁺ population was analyzed. For Bregs, the CD19⁺ CD5⁺ CD1d⁺ population. Fc receptors were blocked using anti-CD16/CD32 Ab. Fluorescence minus one (FMO) controls were included to allow adequate analysis. Samples were acquired on an Attune Nxt Acoustic Focusing Cytometer (Life Technologies) and analyzed using FlowJo software.