Anti mouse cd11c microbeads

Mice were euthanized by CO₂ asphyxiation and lungs were perfused with cold PBS via the heart. Lungs were removed, minced finely with scissors and subjected to enzymatic digestion with 0.5mg/mL Liberase (Roche) for 30 minutes at 37°C as reported previously (Gibbings et al., 2017 (link)). Mediastinal lymph nodes or spleens were removed, minced, and digested by incubation with 2.5mg/mL Collagenase D for 30 minutes at 37°C. Single cell suspensions were prepared by manual homogenization and filtration through a 100 mm nylon filter membrane. Mouse lung macrophages were labeled with anti-mouse MerTK and enriched with anti-biotin microbeads as previously reported. Mouse lymph node and spleen DCs were enriched with anti-mouse CD11c microbeads and spleen monocytes were enriched using anti-mouse CD11b microbeads according to the manufacturer’s protocol (Miltenyi Biotec).

The NKT cell hybridoma 2E10 was cultured as described (25 (link)). Bone marrow-derived DCs from B6 mice were prepared as described (23 (link), 26 (link)), where after 6 days of culture in a complete RPMI-1640 medium (ThermoFisher Scientific) supplemented with 5 ng/mL mGM-CSF (R&D), DCs were purified with AutoMACS and anti-mouse CD11c microbeads (Miltenyi Biotec). Human DCs were prepared as described (27 (link)), where CD14⁺ monocytes were purified from PBMNCs with a MACS LS column and anti-human CD14 microbeads (Miltenyi Biotec) and cultured for 6 days in a DendriMACS GMP medium containing 800 U/mL hGM-CSF and 250 U/mL hIL-4 (all from Miltenyi Biotec). Human umbilical cord blood derived mononuclear cells were prepared by density gradient centrifugation using Ficoll-Paque Plus (GE Healthcare), and NKT cell cultures were performed as reported (23 (link)) with a minor modification, where the culture medium consisted of 50% AIM-V medium (ThermoFisher Scientific), 45% RPMI-1640, 5% heat-inactivated fetal bovine sera (Sigma), 1 × NEAA, 1 mM sodium pyruvate, 55 µM 2-ME, 2 mM l-glutamine, and 100 U/mL penicillin/streptomycin (all from ThermoFisher Scientific) and supplemented with 100 U/mL hIL-2 (Shionogi, Japan).

BMDCs were generated from the femoral and tibial bone marrow cells of female mice as described previously [22 (link)]. Cells were incubated in RPMI 1640 (Sigma-Aldrich, St Louis, MO) supplemented with 10% heat-inactivated fetal calf serum, 100 U/mL of penicillin, 100 μg/mL streptomycin, 100 μM 2-mercaptoethanol, 10 μM Minimum Essential Medium nonessential amino acid solution, and 20 ng/mL of murine GM-CSF (PeproTech, London, United Kingdom) at 37°C in a humidified atmosphere in the presence of 5% CO₂ for 10 days.
Splenic DCs were isolated from mouse spleen as described previously [23 (link)] with slight modifications. Briefly, spleens were injected into, and incubated in RPMI 1640 containing 125 μg/ml Liberase TL (Roche Diagnostics, Mannheim, Germany), 200 μg/ml DNase I (Roche), and 10 mM HEPES at 37°C for 30 min while shaking. Single cells were prepared by passing through a 70-μm cell strainer. After centrifugation at 400 ×g for 5 min, erythrocytes were then lysed using ACK buffer.
CD11c⁺ cells were isolated by using the MACS separation system with anti-mouse CD11c MicroBeads (#130-052-001) and an autoMACS (all from Miltenyi Biotech, Tubingen, Germany).
LPS (#L3024) and trichostatin A (TSA) (#T8552) were purchased from Sigma-Aldrich. HDAC inhibitors, MS-275 (#S1053), Droxinostat (#S1422), and MC1568 (#S1484) were purchased from Selleckchem (Houston, TX).

BMDCs were generated from the femoral and tibial bone marrow cells of female mice as described previously49 . Cells were incubated in RPMI 1640 (Sigma-Aldrich, St Louis, MO) supplemented with 10% heat-inactivated fetal calf serum, 100 U/mL of penicillin, 100 μg/mL streptomycin, 100 μM 2-mercaptoethanol, 10 μM Minimum Essential Medium nonessential amino acid solution, and 20 ng/mL of murine GM-CSF (PeproTech, London, United Kingdom) for 10 days. CD11c⁺ cells were isolated by using the MACS separation system with anti-mouse CD11c MicroBeads (#130-052-001) and an autoMACS (all from Miltenyi Biotech, Tubingen, Germany). Splenic DCs were also isolated from mouse spleen by using the MACS separation system with anti-CD11c MicroBeas50 (link).
Mouse DC line JAWSII (CRL-11904, American Type Culture Collection, Manassas, VA) were maintained in MEM-α (GIBCO) supplemented with 20% FCS, 4 mM L-glutamine, 1 mM sodium pyruvate, 100 U/mL of penicillin, 100 μg/mL streptomycin, and 5 ng/mL of murine GM-CSF. All cells were incubated at 37 °C in a humidified atmosphere in the presence of 5% CO₂.
LPS (#L3024) and poly I:C (#P0913) were purchased from Sigma-Aldrich. CpG-ODN (#tlrl-1826) was obtained from InvivoGen (San Diego, CA).