Nk1.1 apc pk136

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.

Single-cell suspensions from mouse subcutaneous tumors were prepared as described above, counted, and resuspended in PBS at a concentration of 1×10⁷ live cells/mL. One hundred microliters of each sample was plated for cell staining. Surface staining was performed at room temperature for 30 min, and intracellular staining was performed using a Foxp3-transcription factor staining kit (eBioscience). Live/dead cell discrimination was performed using Fixable Viability Stain 520 (BD Biosciences). Anti-mouse antibodies against the following antigens were used for flow cytometry: CD3 PerCP-CY5.5 (17A2, BioLegend, 1:100), CD45 PerCP (30-F11, BioLegend, 1:200), NK1.1 APC (PK136, BD Biosciences, 1:50), Foxp3 PE (MF23, BD Biosciences, 1:100), Ly6G PE (1A8, BioLegend, 1:100), Ly6C APC (HK1.4, BioLegend, 1:100), CD11b BV421 (M1/70, BioLegend, 1:100), CD11b PerCP-CY5.5 (HL3, BioLegend, 1:100), F4/80 APC (T45–2342, BD Biosciences, 1:50), CD25APC (3C7, BioLegend, 1:100), CD4 APC (GK1.5, BioLegend, 1:100), and CD8a PE (53-6.7; BioLegend, 1:100). All flow cytometry analyses were performed using a Fortessa flow cytometer (BD Biosciences) and analyzed using FlowJo software (TreeStar).