Apc conjugated anti ly6c

Aortas were dissected, minced using scissors and enzymatically digested with 200 U/mL Liberase (Roche) and 40 U/mL DNase I (Sigma Aldrich) in HBSS plus 5% FCS for 1 h at 37°C. Cells were filtered through a 40 μm nylon strainer, washed with HBSS plus 5% FCS, collected by centrifugation at 400 g for 5 min at 4°C and then suspended in FACS buffer (PBS plus 0.2% FCS plus 1 mM EDTA). Murine Fc receptors were blocked using anti-CD16/32 antibodies (BioLegend) for 10 min on ice. Violet 510 Viability Dye (Cell Signaling Technology) was used to discriminate between live and dead cells. The cells were stained with the following antibodies for 30 min at 4°C: PerCP-Cy5.5-conjugated anti-CD45, APC-Cy7-conjugated anti-CD11b, FITC-conjugated anti-Ly6G, PE-Cy7-conjugated anti-F4/80, and APC-conjugated anti-Ly6C (all purchased from BioLegend). Flow cytometric analysis was performed using FACSCanto II flow cytometer (BD Biosciences) and DIVA Software (BD Biosciences).

Isolated cells were resuspended in 2% FBS/PBS solution and blocked with FcBlock on ice. Immune cell subpopulations were identified by staining for 20 minutes at room temperature with the following antibodies: Alexa Fluor 647–conjugated anti-CD45 (1:100; eBioscience; catalog 17-0112); FITC-conjugated anti-CD11b (1:100; eBioscience; catalog 17-0112); phycoerythrin-(PE)–conjugated anti-F4/80 (1:100; eBioscience; catalog 12-4801 or 53-4801); BV421–conjugated anti-Ly6G (1:100; BioLegend; catalog 141709); APC-conjugated anti-Ly6C (1:20; BioLegend; catalog 117326); BV421-conjugated anti-CD64 (1:100; BioLegend, catalog 139309); Alexa Fluor 700–conjugated anti-CD206 (1:100; BioLegend, catalog 141734); PE-Cy7–conjugated anti-MerTK (1:100; eBioscience, catalog 25-5751-82); BV421-conjugated anti-CD120a (1:100; R&D Systems, catalog FAB5538P); APC-Cy7–conjugated anti-Csf1r (1:100; BioLegend; catalog 135531, anti-Tmem173 (1:200; Abcam; catalog ab92605); and Alexa Fluor 488– (1:400; Invitrogen; catalog A21206) and APC-conjugated anti-CD120b (rat, 1:100; eBioscience; catalog 45-5932). 7AAD was added prior to cell analyses to exclude dead cells. Flow cytometric analyses and cell sorting were performed using a BD FACSAriaIII flow cytometer (BD Biosciences) and FlowJo software (Tree Star).

Zymosan A (ZyA; Alfa Aesar), DMSO (Tocris Bioscience), Rapamycin (Adipogen), 6-Diazo-5-oxo-L-norleucine (DON; Sigma-Aldrich), Torin1 (Tocris Bioscience), compound 968 (C968; Calbiochem) were purchased. In vitro experiments used the carboxyfluorescein diacetate succinimidyl ester (CFSE) cell proliferation kit (Invitrogen), RPMI 1640 (Wako Pure Chemical Industries), fetal bovine serum (FBS; MP Biomedicals), 1% penicillin-streptomycin (Lonza Walkersville), and recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF; PeproTech). For flow cytometry analysis of myeloid cells, we used FITC-conjugated anti-Gr1 (BD PharMingen), FITC-conjugated anti-Ly6G (BD PharMingen), PerCP-conjugated anti-CD11b (BioLegend), APC-conjugated anti-Ly6C (BioLegend), APC-conjugated anti-CD11c (eBioscience), and PE-conjugated anti-F4/80 (eBioscience). For the flow cytometry analysis of lymphocytes, we used FITC-conjugated anti-CD4 (eBioscience), APC-conjugated anti-Rorγt (eBioscience), PE-conjugated anti-Foxp3 (eBioscience), and 7-AAD Viability Staining Solution (eBioscience). For the Western blotting analysis, anti-phospho-p70 S6 kinase (Thr389) (Cell Signaling, #97596 S), anti-p70 S6 kinase (Cell Signaling, #9202), anti-phospho-Akt (Ser473) (Cell Signaling, #4060), anti-Akt (Cell Signaling, #2920), and anti-β-actin antibodies (Sigma-Aldrich) were purchased.