5x106 cells were lysed in RIPA buffer in the presence of a protease inhibitor cocktail (ThermoFisher) for 30 min at 4°C. Protein concentration was determined by Bradford assay (ThermoFisher). Dendritic cells (DC) were derived from CD14pos peripheral blood monocytes (CD14 positive selection kit, Miltenyi) that were cultured for five days with 20 ng/ml recombinant GM-CSF (Miltenyi) and 50 ng/ml IL-13 (Sanofi-Aventis donation). Equal amounts of protein were resolved on 4-20% Mini Protean gradient gels (Bio-Rad Laboratories) then transferred to nitrocellulose membranes (ThermoFisher). Protein expression was analyzed using anti-AXL (C89E7, Cell Signaling Technology) or anti-DC-SIGN (D7F5C, Cell Signaling Technology) rabbit mAbs followed by HRP-conjugated Goat Anti-Rabbit IgG secondary antibody (Jackson ImmunoResearch). Blots were developed by chemiluminescence assay (Bio-Rad Laboratories). Anti-β-actin (mouse mAb, Sigma Aldrich) was used to confirm protein loading.
Recombinant gm csf
Manufactured by Miltenyi Biotec
Sourced in Japan, France
Recombinant GM-CSF is a cytokine that stimulates the production and function of granulocytes and monocytes. It is produced using recombinant DNA technology.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions)
Chemiluminescence assay, by Bio-Rad (1 mentions)
Bradford assay, by Thermo Fisher Scientific (1 mentions)
Cd14 positive selection kit, by Miltenyi Biotec (1 mentions)
Anti axl c89e7, by Cell Signaling Technology (1 mentions)
Hrp conjugated goat anti rabbit igg secondary antibody, by Jackson ImmunoResearch (1 mentions)
Mouse anti β actin mab, by Merck Group (1 mentions)
Stable glutamine, by Capricorn (1 mentions)
Interleukin 4 (il 4), by STEMCELL (1 mentions)
Lipopolysaccharides (lps), by Nacalai Tesque (1 mentions)
Penicillin streptomycin, by Nacalai Tesque (1 mentions)
Mojosort magnetic cell separation system, by BioLegend (1 mentions)
Lymphoprep, by Axis-Shield (1 mentions)
Ifn γ, by Miltenyi Biotec (1 mentions)
Fetal bovine serum (fbs), by Capricorn (1 mentions)
Zymosan, by InvivoGen (1 mentions)
Anti cd11c microbeads, by Miltenyi Biotec (1 mentions)
Lps eb, by InvivoGen (1 mentions)
Gm csf, by InvivoGen (1 mentions)
4 protocols using recombinant gm csf
1
Quantifying Dendritic Cell Protein Expression
2
Isolation and Polarization of M1/M2 Macrophages
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Thirty ml of peripheral blood was withdrawn from three groups of CAD patients. Peripheral blood mononuclear cells (PBMCs) were isolated by collecting the buffy coat generated after Lymphoprep™ (Axis Shield, Oslo, Norway) density centrifugation. Mojosort™ magnetic cell separation system (Biolegend, San Diego, California) was employed to further isolate monocytes from the pooled PBMCs. Next, monocytes were re-suspended (1 × 106/ml) in RPMI1640, with stable glutamine (Capricorn Scientific, Germany) and seeded into 25 cm2 tissue culture flask (SPL Life Sciences, Korea). Monocytes were allowed to adhere at 37 °C, 5% CO2 for 3 h. Non-adherent cells were washed off using RPMI 1640 with stable glutamine media. The adherent monocytes were cultured for 5 days in RPMI 1640 with stable glutamine media supplemented with 10% heat-inactivated fetal bovine serum (Capricorn Scientific, Germany), 1% penicillin-streptomycin (Nacalai Tesque, Japan), and 20 ng/ml recombinant GMCSF (Miltenyi Biotec, Germany) for M1 macrophage or 10 ng/ml recombinant MCSF (Gold Biotechnology, Missouri) to generate M2 macrophages. After day 5, M1 macrophages were polarized with 10 ng/ml LPS (Nacalai Tesque, Japan) and 20 ng/ml IFN-γ (Miltenyi Biotec, Germany) for 2 days. Meanwhile, 20 ng/ml IL-4 and 20 ng/ml IL-13 (Stemcell Technologies, Canada) were added into culture media to polarize M2 macrophages.
3
Stimulation of Bone Marrow Derived Cells
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Cell suspensions from bone marrow of the indicated genotypes including WT, Myd88−/− (Adachi et al., 1998 (link)), CD11cΔSyk (Iborra et al., 2012 (link)), Fcer1g−/− (B6; 129P2-Fcer1gtm1Rav/J), Clec7a−/− (Marakalala et al., 2013 (link)), Clec4n−/− (Saijo et al., 2010 (link)) and Clec4e−/− (Wells et al., 2008 (link)) were cultured on tissue culture flasks (Falcon Products) in the presence of 20 ng/mL recombinant GM-CSF (Miltenyi Biotec). GM-CSF BM-derived cells were collected on day 6, purified by positive selection with anti-CD11c-microbeads (Miltenyi Biotec) and plated 16 h before stimulation.
In the indicated experiments, GM-CSF BM-derived CD11c+ cells (GM-BM, 2x106/mL) were stimulated with LPS EB (100 ng/mL, InvivoGen), plated TDB (1 μg/well, InvivoGen), zymosan (10 μg/mL, InvivoGen) or by co-culture with the indicated ratio of total microbiota from SPF mice, quantified by the optical density at 600 nm (OD600nm) together with quantification by flow cytometry, in the presence of 100 U/mL Penicillin and 100 μg/mL Streptomycin. Activation of GM-BMs was assessed by quantifying the upregulation of MHCII, CD40, CD86, CCR7 or Syk phosphorylation.
For some experiments, GM-BMs were FACS-sorted based on the expression of MERTK and CD115 into conventional DCs (GM-DCs) and monocyte-derived macrophages (GM-Macs) as previously described (Helft et al., 2015 (link)).
In the indicated experiments, GM-CSF BM-derived CD11c+ cells (GM-BM, 2x106/mL) were stimulated with LPS EB (100 ng/mL, InvivoGen), plated TDB (1 μg/well, InvivoGen), zymosan (10 μg/mL, InvivoGen) or by co-culture with the indicated ratio of total microbiota from SPF mice, quantified by the optical density at 600 nm (OD600nm) together with quantification by flow cytometry, in the presence of 100 U/mL Penicillin and 100 μg/mL Streptomycin. Activation of GM-BMs was assessed by quantifying the upregulation of MHCII, CD40, CD86, CCR7 or Syk phosphorylation.
For some experiments, GM-BMs were FACS-sorted based on the expression of MERTK and CD115 into conventional DCs (GM-DCs) and monocyte-derived macrophages (GM-Macs) as previously described (Helft et al., 2015 (link)).
4
Monocyte-Derived Dendritic Cell Isolation and Activation
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Peripheral blood mononuclear cells (PBMC) were isolated from buffy coats (blood donors from EFS, Toulouse) by Ficoll separation (PAA). Monocytes were isolated from PBMC with the use of CD14 microbeads and a magnetic bead separation system (Miltenyi, Paris, France). Monocytes were maintained for 5 days in RPMI medium supplemented with 10% FCS, 1% penicillin-streptomycin, 20 ng/mL recombinant GM-CSF (Miltenyi, Paris, France) and 50 ng/mL IL-13 (donated by Sanofi-Aventis). On day 5, non-adherent moDC were recovered and stimulated for 24 h with either S. aureus or S. epidermidis secretome at 5% (v/v), or LPS at 100 ng/mL or ADE (lg/mL) as indicated. Cells were stained and submitted to flow cytometry analysis on a FACSCalibur cytometer (BD Biosciences, Le Pont de Claix, France) as detailed in the flow cytometry analysis section.
cells were isolated from PBMC with the use of naive CD4 þ T cell isolation kit II and CD4 T cell isolation kit II (Miltenyi, Paris, France). For proliferation assays, CD4 þ T cells were stained with CFSE (Invitrogen, Waltham, MA) and cocultured with allogeneic moDC as indicated for 5 days in 96-well plates at a ratio of 1 stimulator to 10 T cells. Proliferation (%) was assessed by flow cytometry.
cells were isolated from PBMC with the use of naive CD4 þ T cell isolation kit II and CD4 T cell isolation kit II (Miltenyi, Paris, France). For proliferation assays, CD4 þ T cells were stained with CFSE (Invitrogen, Waltham, MA) and cocultured with allogeneic moDC as indicated for 5 days in 96-well plates at a ratio of 1 stimulator to 10 T cells. Proliferation (%) was assessed by flow cytometry.
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