CD11c+ splenocytes were enriched to 80–85% purity by positive selection (anti-CD11c MACS microbeads) following manufacturer instructions (Miltenyi Biotec, Auburn, CA). CD11c+ splenocytes were then incubated overnight at 37°C in complete medium with or without 10 μM NP311−325 peptide. After overnight incubation, cells were washed once and added to single cell suspensions of lung cells.
Anti cd11c macs microbeads
Manufactured by Miltenyi Biotec
Sourced in Germany
Anti-CD11c MACS microbeads are designed for the magnetic separation of CD11c-positive cells from heterogeneous cell populations. The microbeads are conjugated with an antibody specific for the CD11c surface antigen, which is expressed on dendritic cells and certain subsets of monocytes and macrophages.
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4 protocols using anti cd11c macs microbeads
1
Enrichment and Stimulation of CD11c+ Splenocytes
2
Isolation and Culture of Bone Marrow Stromal Cells
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BM cells from long bones were cultured for 7 days in α-MEM medium containing 10% FBS, 100 mg/mL of penicillin, and 100 IU/mL of streptomycin, to allow the proliferation of stromal cells (SCs). After removing non adherent cells, adherent macrophages were eliminated by positive immunoselection using anti-CD11c MACS Microbeads (Miltenyi Biotech). The remaining adherent cells were defined as SCs as they express alkaline phosphatase (ALP), type-I collagen, and Runx2, and have the capacity to form mineralization nodules when further cultured under mineralizing conditions.
3
Isolation and Culture of Mouse Bone Marrow-Derived Dendritic Cells
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Primary bone marrow DCs were obtained from mouse bone marrow precursors as previously described,8 (link),19 (link) with minorc modifications. DCs were cultured at a starting concentration of 2.0×106 cells/mL in round-bottomed 6-well plates with RPMI 1640 supplemented with 10% FCS (Gibco, Waltham, MA, USA). Cells were cultured in a humidified chamber at 37°C and 5% atmospheric CO2. After incubation for 24 h, the medium with nonadherent cells was replaced with RPMI 1640 medium supplemented with 10% FCS, 20 ng/mL GM-CSF (R&D; BJ2313011), and rmIL-4 (R&D; BC612121). The culture medium was removed and replenished with an equal volume of fresh medium every 2 days. From day 6 onward, the nonadherent DCs were harvested and used for subsequent experiments. DCs were purified by positive selection with anti-CD11c MACS MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany), following the manufacturer’s protocol.
4
Laminin Substrates for Dendritic Cell Adhesion
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Purified human placental laminin 51147 (link); recombinant laminin α5 domain IVa31 (link); mini-laminin 511 composed of the C-terminal sequences of laminin α5, β1 and γ1 chains (Takaa Bio, USA)54 (link),55 (link); human serum fibronectin56 (link) were employed as substrates. Substrates were plated onto 24-well plates at 10 μg/ml by overnight incubation at 4°C, non-specific binding was blocked for 30 minat 37°C using 1% BSA in PBS. To isolate DCs, lymph nodes were dissected into small pieces and digested in HEPES buffer containing 2% FCS, 2 mg/ml Collagenase D (Sigma) and 20 μg/ml DNase I (Sigma) at 37°C for 30 minutes. After filtering through a 100 μm cell strainer, DCs were isolated using anti-CD11c MACS microbeads (Miltenyi). 2 x106 cells were added/24-well and incubated at 37°C for 90 mins; after washing with PBS, adherent cells were analysed by flow cytometry for CD11c+Cd11b+ cells. For inhibition studies, cells were incubated with 20 μg/ml GoH3 antibody or isotype control.
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