Xcr1 apc

Cells were incubated 10 min with the TruStain fcX (clone 93) washed and incubated with the antibodies during 20 minutes followed by two washes with PBS. Monoclonal antibodies specific for mouse molecules were purchased from Biolegend: CD3-FITC (clone 17A2), CD3-APC (clone 17A2), CD3-PerCp/Cy5.5 (clone 17A2), CD8-Brillant Violet 421 (clone 53-6.7), CD45-PE (clone 30-F11), CD45-PErCP(clone 30-F11), CD45.1-PE/Cy7 (clone A20), CD45.1-FITC (clone A20), CD103-APC (clone 2E7), CD103-PerCP (clone 2E7), CD69-APC/Cy7 (clone H1.2F3), CD69-APC (clone H1.2F3), CD44-PerCP (clone IM7), IFN-γ-PE (clone XMG1.2), IFN-γ-APC (clone XMG1.2), TNF-α-APC/Cy7 (clone MP6-XT22), CD11b-FITC (clone M1/70), CD207-PE (clone 4C7), XCR1-APC (clone ZET), XCR1-PerCP-Cy5.5 (clone ZET), CD11c-PE/Cy7 (clone N418), MHCII-APC/Cy7 (clone M5/114.15.2), CD24-PerCP-Cy5.5 (clone M1/69), CD80-APC (clone 16-10A1), CD80-PE/Cy7 (clone 16-10A1), CD86 Brilliant Violet 421 (clone GL-1), CCR7-PE/Cy7 (clone 4B12), IL-2-PE/Cy7 (clone JES6-5H4) IL-12/23-APC (clone C15.6), granzyme B-APC (clone GB11) and viability dye Zombie Aqua (ref 423101). Samples were acquired in a FACSCanto II cytometer (BD Bioscience) and data were analyzed using FlowJo version X.0.7 (Tree Star, Inc.). Gating strategies for all flow cytometry experiments are shown in Supplementary Fig. 4.

Bone marrow cells were isolated by flushing marrow from tibias and femurs using a 27-gauge needle and PBS. Cells were cultured at 1.5 x 10⁶ cells/ml for 7 days in RPMI 1640 media (Wisent) supplemented with 10% FBS (Wisent), 50 μM β-mercaptoethanol, antibiotic/anti-mycotic (Wisent) and 100 ng/ml FMS-like tyrosine kinase 3 ligand (FLT3L) (Peprotech, catalog no. 250-31L). On day 7, 10 ng/ml of Granulocyte-macrophage colony-stimulating factor (GM-CSF) (Peprotech, catalog no. 315–03) was added to the culture. For the stimulation of FLT3L-BMDCs, 10⁴ live or an antigenic preparation (obtained by two cycles of freeze and thaw) of B16F10 or LLC cells per million of DCs were added. In some stimulations, DCs were segregated from live cancer cells with a 0.4 μm cell culture insert (Falcon). For transfer experiments, FLT3L-BMDCs were stimulated on day 7 with GM-CSF and on day 9 with live B16F10 cells and harvested on day 10. Before the injection, XCR1⁺ FLT3L-BMDCs were isolated using XCR1-APC (Biolegend) and EasySep™ Mouse APC Positive Selection Kit II (Stemcell).