Ebioscience fixable viability dye efluor 506

Cells were suspended in PBS containing 2% FBS and stained with fluorescent-labeled anti-mouse surface molecule antibodies: CD4 (RM4-5)/PerCP, CD25 (PC61)/BV421, CD8 (53-6.7)/FITC, TNFR2 (TR75-89)/PE, and CD90.2 (30-H12)/PE (BioLegend, San Diego, CA). Live cells were stained by eBioscience Fixable Viability Dye eFluor 506 (Thermo Fisher Scientific). Anti-mouse CD16/CD32 (2.4G2) (BD Biosciences) was used for Fc blocking. For intracellular staining, anti-Foxp3 (FJK-16s)/APC (Thermo Fisher Scientific), anti-Foxp3 (ME-14)/BV421, anti-IL-4 (11B11)/PE, anti-Ki-67 (16A8)/PE (BioLegend), anti-IL-17(TC11-18H10)/PE (BD Biosciences), anti-phospho-IκB alpha (Ser32, Ser36) (RILYB3R)/eFluor 660 (Thermo Fisher Scientific), and anti-phospho-IKKα/β (Ser176/180) (16A6) /PE (Cell Signaling Technology) were used after fixation and permeabilization with an eBioscience Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific) according to the manufacturer’s protocol. Isotype controls matched for each antibody were used. For human molecule staining, anti-CD4 (RPA-T4)/APC (BioLegend), anti-Foxp3 (PCH101)/PE (eBioscience), and anti-TNFR2 (hTNFR-M1)/BV421 (BD Biosciences) were used. FCM was performed using a CytoFLEX cell analyzer (Beckman Coulter, Brea, CA). Data were analyzed using FlowJo software (BD Biosciences).

Therapeutic vaccination was performed as described before^{82 (link)}. Spleen from C57BL/6J mice was isolated, cut into small pieces and incubated with the mix of Liberase™ TM Research Grade (5401127001, Roche) DNAse I (10104159001, Roche) for 30 min at 37 ^oC according to previously published protocol⁸³ . Afterward, the single-cell suspension was washed in 1xHBSS (14175-053, Gibco) and red blood cells were removed by 3 min incubation with ACK buffer (A1049201, Gibco). After that, the cell suspension was incubated with TAMRA labeled BM1-OVA cells killed by necroptosis or ferroptosis for 4 h at 37 °C. Following the staining with antibodies for XCR1-BV650 (#148220, BioLegend, 1:200) and CD11c-BV711 (#563048, BD Pharmingen, 1:200) as well as cell death marker eBioscience™ Fixable Viability Dye eFluor™ 506 (1/1000, #65-0866-14, ThermoFisher). CD11c⁺XCR1⁺TAMRA⁺ cells were sorted using FACS. The obtained population was centrifuged, resuspended in 1xHBSS and 5 × 10⁵ cDC1 carrying ferroptotic or necroptotic cells were injected intradermally in C57BL/6J mice bearing B16-OVA subcutaneous tumors. The growth of the tumor was monitored every 3–4 days using an electric caliper. The size of the tumor was determined by the formula: (3.14 × Width × Length × Depth)/6.

Cells were incubated with Fc Block (CD16/32, BD Biosciences, San Jose, CA), stained with antibodies, and then fixed with 2% PFA. Samples were acquired on the FACS Canto II (BD Biosciences) and analyzed using FlowJo (TreeStar, Ashland, OR). Dead cells were excluded using the eBioscience Fixable Viability Dye eFluor® 506 (Thermo Fisher Scientific, Waltham, MA). The following antibodies were used: anti-CD8 (53-6.7) and anti-TCR-γδ (ebioGL3) from Thermo Fisher Scientific; anti-CD4 (GK1.5), anti-CD19 (6D5), anti-CD11b (M1/70), and anti-CD45 (30-F11) from BioLegend, San Diego, CA; and anti-TCR-β (H57-597) from BD Biosciences. APC-conjugated mouse CD1d tetramers loaded with glycolipid PBS-57 (CD1d-tet) and an unloaded tetramer comprised of only the glycolipid PBS57 were obtained from the tetramer facility of the National Institutes of Health (NIH).

BAL-derived cells were labeled for anti-mouse CD11c-phycoerythrin (PE, clone HL3, BD Biosciences) and anti-mouse F4/80-allophycocyanin (APC, clone BM8, BioLegend) antibodies. Peritoneum-derived cells were labeled for anti-mouse F4/80-APC and anti-mouse CD11b-PE- Cyanine7 (PE-Cy7, clone M1/70, eBioscience).
The FcR Blocking Reagent (Miltenyi Biotec) was used to increase specificity by preventing non-speciic binding of antibody conjugates. To exclude dead cells, eBioscience^™ Fixable Viability Dye eFluor^™ 506 (Thermo Fischer Scientific) was applied based on the manufacturer’s instructions.
The CD11c-F4/80 double-positive alveolar macrophages and F4/80hi-CD11bhi large peritoneal macrophages were sorted.
The flow cytometry analysis and cell sorting were performed by BD FACSAria^™ III (BD Biosciences) using BD FACSDiva Software 6.0 (BD Biosciences). Flow cytometry data analysis was performed with FlowJo v10.8 (BD Biosciences).