Percp cy5.5 cd11b

Murine: I-A/I-E-BV421, B220-BV605, F4/80-BV711, Sirpα-PE-Cy7, CD45-FITC, TCR Vβ5.1, 5.2-APC, BV421-CD4, PE-Cy7 CD8a, PerCP-Cy5.5-CD11b, CD40-BV786, H2K^b/H-2D^b-Alexa647 (BioLegend); TLR7-PE, CD24-BUV395, CD80-BUV737, F4/80-BV421, CD11b-APC (BD); and eBioscience Fixable Viability Dye eFluor 780 (Thermo Fisher Scientific). Western blotting: TRIF (Genentech,), MYD88 (Abcam), β-actin (Cell Signaling Technologies). Human: CD64-APC, CD80-BV786 (BD); CD163-FITC, B2M-PE, CD209-APC (DC-SIGN); CD86-PE, CD80-PE-Cy7 (BioLegend); and CD14-PerCP-eFluor 710, CD81-FITC (Invitrogen). LIVE/DEAD Fixable Aqua Dead Cell dye and LIVE/DEAD Fixable Blue Dead Cell Stain Kit for UV excitation were from Thermo Fisher Scientific.

Cardiac leukocyte isolation was performed as previously described (Eberhardt et al., 2019 (link)). Briefly, hearts were perfused with phosphate-buffered saline (PBS) and disaggregated mechanically and enzymatically with 0.2% trypsin solution (Gibco). The digested tissue was pressed through a 70 μm cell strainer (BD Falcon), and cells were isolated by 35 and 70% bilayer Percoll (GE Healthcare) density gradient centrifugation. Viable cell numbers were determined by trypan blue dye exclusion using a Neubauer chamber, and absolute cell number was obtained corresponding to the whole heart. Cells were stained with the following antibodies: anti-mouse fluorescein isothiocyanate (FITC)-CD3, APC-Cy7-CD4, PE-Cy7-CD8, PE-CD19, PerCP-Cy5.5-CD11b, FITC or APC-Cy7-CD11c, PE-F4/80, PE-Cy7-CD206, APC-Ly6G, and APC-Cy7-Ly6C (all from BioLegend). Stained samples were acquired using FACS Canto I and II cytometers (Becton Dickinson), and the data were analyzed using FlowJo software (Tree Star). Non-specific fluorescence was determined using isotype controls.

Nr-CWS was supplied by Greatest Biopharma Limited Company (purity>98%). Fluorescein conjugated anti-mouse antibodies APC-NK1.1, FITC-NK1.1, APC/Cy7-CD3ε, PE/Cy7-CD3ε, APC-CD4, PE-CD8α, PerCP/Cy5.5-CD11b, PE-CD27, FITC-TNF-α, and PerCP/Cy5.5-IFN-γ from BioLegend, and PerCP/Cy5.5-CD69, FITC-FasL, PE-TRAIL, PE-Perforin, and FITC-Granzyme-B from eBioscience were used in this study. TUNEL assay kit and MTS Cell proliferation, cytotoxicity or chemosensitivity assays kit (cat # 3580) were obtained from Promega. The LEGENDplex™ Multiplex mouse cytokine kit was provided by BioLegend. EasySep™ mouse NK cell isolation kit was from STEMCELL Technologies Inc. Recombinant murine IL-2 was from PeproTech Inc.

Cell suspensions were stained with appropriate antibodies for 30 min on ice. The commercial antibodies used in this study included anti-mouse FITC-CD45 (BioLegend, CA, USA, #103108; 1 µg per 10⁶ cells), PE-CLEC2 (BioLegend, CA, USA, #146104; 1 µg per 10⁶ cells), BV421-F4/80 (BioLegend, CA, USA, #123137; 1 µg/10⁶ cells), Pe/Cy7-CD64 (BioLegend, CA, USA, #128016; 1 µg per 10⁶ cells), Percp/Cy5.5-CD11b (BioLegend, CA, USA, #101228; 1 µg per 10⁶ cells), APC/Cy7-PirB (R&D, CA, USA, #FAB2754S; 5 µg per 10⁶ cells), APC-Ly6C (BioLegend, CA, USA, #128016; 1 µg per 10⁶ cells), APC-Ly6G/Ly-6C (Gr-1) (BioLegend, CA, USA, #108411; 1 µg per 10⁶ cells), PerCP-CD19 (BioLegend, CA, USA, #115531; 1 µg per 10⁶ cells), APC-CD3 (BioLegend, CA, USA, #100235; 1 µg per 10⁶ cells), APC-CD206 (BioLegend, CA, USA, #141707; 2 µg per 10⁶ cells), and FITC-CD11c BioLegend, CA, USA, #117305; 1 µg per 10⁶ cells); and anti-human PE-CD14 (BD Biosciences Pharmingen, USA, #555398; 20 µl per 10⁶ cells), Mouse anti-human LILRB2 (R&D, R&D Systems, MN, USA, #MAB2078; 0.25 µg per 106 cells), and Goat Anti-Mouse FITC-IgG (Servicebio, Wuhan, China, #SF131; 1:200 dilution). All antibodies were diluted according to the manual from the manufacturer’s website. Dead cells and doublets were removed by dead-cell dye staining (Zombie Aqua Fixable Viability Kit, BioLegend, CA, USA, #B297827).

Bone marrow and spleen of a transplant recipient mouse were harvested, and single cell suspensions were prepared as previously described34 (link). Briefly, the femurs and tibias were dissected using sterile technique, flushed with PBS/5% FBS, and cells were passed through a 70 μm strainer. Spleens were dissected cleanly using sterile technique, placed on a 70 μm strainer and using the plunger end of a syringe, gently passed through the strainer. Red blood cells were lysed using RBC lysis buffer (Biolegends, San Diego, California). Cells were resuspended in PBS/5% FBS, and then counted using a Cellometer Mini cell counter (Nexcelom, Lawrence, MA). One million cells of each sample were then distributed into microcentrifuge tubes and labelled with fluorochrome-conjugated antibodies APC/Cy7-B220, FITC-Ter119, PE/Cy7-Gr1, and PerCP/Cy5.5-CD11b (all antibodies were purchased from Biolegends and added at predetermined optimum concentrations). Immunophenotyping was performed in the UVA Flow Cytometry core lab using a Fortessa cytometer, and data were analyzed using the FlowJo program (FlowJo LLC, Ashland, Oregon).