Mhc class 2 m5 114.15.2

Mouse BMDCs were dispensed into 12-well plates at a concentration of 5 × 10⁵ cells/well and incubated with rLdpA (1 μg/mL), chitin (50 μg/mL), rLdpA–chitin complex (rLdpA, 1 μg/mL; chitin, 50 μg/mL), or lipopolysaccharide (LPS, 10 ng/mL) (eBioscience, San Diego, CA, USA) for 16 h. Cells were suspended in FACS buffer (2% heat-inactivated FBS and 1 mM EDTA in PBS, pH 7.4). Fc receptors were blocked with anti-mouse CD16/32 mAb (93; eBioscience). Cells were stained with a mixture of fluorochrome-conjugated monoclonal antibodies (mAbs) to CD11c (N418; eBioscience), MHC class II (M5/114.15.2; Miltenyi Biotec), CD40 (FGK45.5; Miltenyi Biotec), CD80 (16-10A1; Miltenyi Biotec), and CD86 (PO3.3; Miltenyi Biotec) in FACS buffer. CD11c⁺ cells were gated, and MHC class II, CD40, CD80, and CD86 cell-surface expression were measured by flow cytometry (BD FACSVerse; BD Biosciences). Data were analyzed using FlowJo version 10 (BD Biosciences).