Anti cd24 m1 69

Bone marrow cells were harvested by flushing tibia and femur with RPMI medium with 10% FCS. The cell suspension was filtered through a 75 μm Nylon cell strainer, seeded at a concentration of 2 × 10⁶ cells/ml, 5 ml/well in a 6-well plate. Then, 0.1 μg/ml of Flt3L (Peprotech, NJ) was added and the cells were incubated for 9 days at 37°C 5% CO₂ (29 (link)). Semi-adherent cells were subsequently harvested and analyzed by flow cytometry after staining with anti-CD45R/B220 (RA3-6B2, Tonbo Biosciences), anti-CD11c (N418, Tonbo Biosciences), anti-CD11b (M1/70, Tonbo Biosciences), and anti-CD24 (M1/69, BioLegend) for 20 min on ice. For staining with mCherry vaccibodies, the cells were subsequently incubated with purified vaccibody proteins at a concentration of 20 μg/ml for 25 min on ice.

The following antibodies were used in this study: anti-CD3 (G4.18), anti-CD44 (IM7), anti-CD62L (MEL-14), anti-IL-2 (JES6-5H4) and anti-CD5 (53-7.3) were purchased from BD Bioscience. Anti-CD4 (RM4-5), anti-CD8 (53-6.7), anti-TCRβ (H57-597) and anti-B220 (RA3-6B2) were purchased from ebioscience. Anti-IFNγ (XMG1.2), anti-CD69 (H1.2F3) and anti-CD24 (M1/69) were purchased from Biolegend. Anti-HDAC1 (ABE260) and Anti-HDAC2 (3F3) were purchased from Millipore. Anti-rabbit IgG1 (H+L) was purchased from Invitrogen.

DCs from spleens of BALB/c mice were prepared using the GentleMACS dissociator (Miltenyi Biotech) according to the manufacturer's protocol. Briefly, spleens were dissociated in GentleMACS C tubes in medium containing collagenase and DNase, incubated for 15 min at 37 C before adding EDTA at a final concentration of 10 mM. Erythrocytes were lysed by incubation with ACT buffer for 5 min on ice. Finally, cells were filtered through a 75 mm Nylon cell strainer. The following Abs were used for subsequent flow cytometry analysis: anti-CD3e (145-2C11; Tonbo Bio- sciences), anti-CD19 (1D3; Tonbo Biosciences), anti-CD49b (DX5; eBioscience), anti-Ly6G (1A8), anti-CD45R (RA3-6B2; Tonbo Biosciences), anti-MHC-II (M5/114.15.2; BioLegend), anti-CD11c (N418; Tonbo Bio- sciences), anti-CD11b (M1/70; Tonbo Biosciences), and anti-CD24 (M1/69; BioLegend).