Cd3 apc 145 2c11

Spleens were perfused with collagenase D (1 mg/ml) and DNAse I (0.1 mg/ml), cut into small pieces and incubated at 37°C for 45 min. Cell preparations were filtered using 100‐μM cell strainers, and APC populations were enriched using magnetic depletion of B, NK, and T cells (Miltenyi). For experiments that focused only on cDC, cDC were enriched using CD11c‐positive selection (Miltenyi). Cell suspensions were labeled with the following antibodies: CD3‐APC (145‐2C11, 1/300 eBioscience), CD19‐APC (ID3, 1/300 Biolegend), NK1.1‐APC (PK136, 1/300 BD Pharmingen), B220‐BV510 (RA3‐6B2, 1/500, BD Horizon), CD11b PE‐CF594 (M1/70, 1/3000 BD Horizon), CD11c PE‐Cy7 (HL3, 1/400 BD Pharmingen), Ly6C‐PerCP‐Cy5.5 (AL‐21, 1/1000 BD Pharmingen), CD64 BV421 (X54‐5/7.1, 1/300 Biolegend), CD8α APC‐Cy7 (53‐6.7, 1/200 BD Pharmingen), or XCR1‐PE (REA707, 1/100 Miltenyi). Cells were sorted on a BD AriaSorp (BD Biosciences) with purity routinely higher than 95%. Expression of MHC II was assessed in a separate mix since the anti‐I‐A^b AF700 (M5/114, 1/500 BD Pharmingen) is a blocking antibody. For experiments with Karma mice in which cDC1 are Tomato⁺, CD11b‐PerCP‐Cy5 (M1/70, 1/200 BD Pharmingen) was used. All flow cytometry samples were run on a Fortessa (BD Biosciences) and analyzed using FlowJo software.

Blood was collected from tail veins in the presence of 5 mM EDTA, incubated with BD Fc Block for 10 min, and then stained with mAbs (BD Bioscience unless indicated otherwise) specific to CD45-APC-Cy7 (clone 30-F11), Ly6G-eFluor450 (1A8-Ly6g, eBioscience), CD11b-PerCP-Cy5.5 (M1/70), Ly6C-FITC (AL-21), NK1.1-APC (PK136), CD3-APC (145-2C11), CD19-APC (1D3), and TER119-APC (TER-119, eBioscience) for 20 min at room temperature. The samples were then fixed and lysed using BD FACS Lysing solution (BD Bioscience). After fixation, cells were washed and resuspended in 2% FBS/PBS. AccuCheck counting beads (PCB100, Invitrogen, Grand Island, NY, USA) were used for quantification of absolute cell numbers. Multiparameter analysis was performed on a FACSSymphony A3 (BD Bioscience) using the FACSDiva software and data were analyzed using the FlowJo software (version 10, Tree Star Inc., Ashland, OR, USA) as described previously [10 (link)].