Pam3CSK4, phorbol 12-myristate 13-acetate (PMA) and ionomycin were purchased from Sigma Aldrich (St. Louis, MO, USA). Dispase, collagenase, DNase, CellROXTMGreen, FluoReporter™ fluorescein isothiocyanate (FITC), a Protein Labeling Kit, Ovalbmin (OVA), and FITC-conjugated OVA were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Percoll was purchased from GE Healthcare (Chicago, IL, USA). A Cytofix/Cytoperm kit with GolgiStopTM was purchased from BD Bioscience (Franklin Lakes, NJ, USA). Recombinant murine granulocyte macrophage-colony stimulating factor (rmGM-CSF) and recombinant murine interleukin-2 (rmIL-2) were purchased from Peprotech (Rocky Hill, NJ, USA). Anti-CD11c (N418), anti-CD11b (M1/70), anti-CD80 (16-10A1), anti-CD86 (GL-1), anti-MHC-II (I-A/I-E; M5/114.15.2), anti-mouse Ly-6G/Ly-6C (Gr-1) (RB6-8C5), anti-CD45 (30-F11), anti-CD3 (17A2), anti-CD4 (GK1.5), anti-Interferon gamma (IFN-γ) (XMG1.2), anti-IL-17A (BL168), and anti-CD16/CD32 (2.4G2) (93) were purchased from Biolegend (San Diego, CA, USA). Anti-CD4 (GK1.5), anti-IFN-γ (H22), anti-IL4 (BVD6-24G2), anti-IL-17A (17F3) mAb, and anti-Ly-6G (1A8) were purchased from Bio X Cell (West Lebanon, NH, USA). The isotype-matched control for each antibody was purchased from the same company.
Recombinant murine granulocyte macrophage colony stimulating factor rmgm csf
Manufactured by Thermo Fisher Scientific
Sourced in United States
Recombinant murine granulocyte-macrophage colony stimulating factor (rmGM-CSF) is a cytokine that regulates the production and function of granulocytes and macrophages. It is a recombinant form of the mouse GM-CSF protein.
Automatically generated - may contain errors
Lab products found in correlation
Dnase, by Thermo Fisher Scientific (3 mentions)
Recombinant murine interleukin rmil 4, by Thermo Fisher Scientific (2 mentions)
Inverted light microscope, by Olympus (2 mentions)
Collagenase, by Thermo Fisher Scientific (1 mentions)
Dispase, by Thermo Fisher Scientific (1 mentions)
Anti ly6g 1a8, by BioXCell (1 mentions)
Pam3csk4, by Merck Group (1 mentions)
Cellrox green, by Thermo Fisher Scientific (1 mentions)
Protein labeling kit, by Thermo Fisher Scientific (1 mentions)
Fitc conjugated ova, by Thermo Fisher Scientific (1 mentions)
Cytofix cytoperm kit with golgistop, by BD (1 mentions)
Percoll, by GE Healthcare (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Poly 1 c, by InvivoGen (1 mentions)
Antibodies against gapdh and β actin, by Santa Cruz Biotechnology (1 mentions)
Cpg odn 1826, by InvivoGen (1 mentions)
Mhcii, by BioLegend (1 mentions)
Rmil 4, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Pam3csk4, by InvivoGen (1 mentions)
8 protocols using recombinant murine granulocyte macrophage colony stimulating factor rmgm csf
1
Murine Dendritic Cell Isolation and Activation
2
Murine Dendritic Cell Activation Assay
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Recombinant murine granulocyte-macrophage colony stimulating factor (rmGM-CSF) and recombinant murine interleukin 4 (rmIL-4) were purchased from Peprotech (Rocky Hill, NJ, USA). LPS was purchased from Sigma (St. Louis, MO, USA). β-mercaptoethanol was obtained from MP Biomedicals LLC (Solon, OH, USA). Pam3csk4, poly (I:C), and CpG ODN1826 were purchased from InvivoGen (San Diego, CA, USA). FITC-, APC-, and PE-conjugated anti-murine CD4, CD11c, CD40, CCR7, CD80, CD86, and MHC-II antibodies were purchased from BioLegend (San Diego, CA, USA) and eBiosciences (New York, NY, USA). Antibodies against CD11c, TLR4, MyD88, and IκB were purchased from Biosynthesis Biotechnology Co. Ltd. (Beijing, China). Antibodies against p65 and p38 were obtained from Zhongshan Golden Bridge Co. Ltd. (Beijing, China). Antibodies against phospho-p65, phospho-IRF3, phospho-ERK1/2, and IRF3 were from Cell Signaling Technology (Danvers, MA, USA). Antibodies against phospho-p38 and ERK1/2 were purchased from Bioworld Technology (Nanjing, China). Anti-Tmod1 antibody was prepared by AbMax Biotechnology Co., Ltd. (Beijing, China). Antibodies against GAPDH and β-actin were obtained from Santa Cruz Biotech. (Santa Cruz, CA, USA). Tmod1 adenovirus and control adenovirus were constructed by SinoGeneMax Co. Ltd. (Beijing, China).
3
Immunofluorescence Analysis of AANAT Regulation
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The reagents adrenaline, noradrenaline, isoprenaline, propranolol, H-89 (PKA inhibitor), the primary antibodies (anti AANAT: S0564; and anti-phospho AANAT: 0939), the secondary FITC-labelled antibody (S0814), and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich (St. Louis, MO, USA). RPMI 1640 medium and fetal bovine serum were purchased from Gibco-Life Technologies (Grand Island, New York, NY, USA). The NF-κB blockers acetyl-l -leucyl-l -leucyl-l -norleucinal (ALLN) and pyrrolidine dithiocarbamate (PDTC) were purchased from Tocris (Minneapolis, MN, USA). The recombinant murine granulocyte–macrophage colony-stimulating factor (rmGM-CSF) were obtained from Peprotech (Rocky Hill, NJ, USA). All reagents were of analytical grade.
4
Chitosan Preparation and Cell Culture
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Chitosan was obtained from Sigma-Aldrich (St. Louis, MO, USA), Acros Organics (Bridgewater, NJ, USA), AK Scientific (Union City, CA, USA), MP Biomedicals (Solon, Ohio, OH, USA), Spectrum Chemical Mfg Corp. (New Brunswick, NJ, USA) and Primex (Siglufjordor, Iceland). Purified, low endotoxin Chitosan (<0.01 EU/mg) was obtained from the University of Arkansas Biologics Center (UABC) (Fayetteville, AR, USA). Sodium hydroxide was purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Hydrochloric acid and acetic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Unless otherwise specified, Chitosan solutions were prepared by dissolving Chitosan in 0.1 M HCl and adjusted to pH 6.0 using NaOH.
Cell culture media components including Dulbecco’s modified Eagle’s medium (DMEM), Roswell Park Memorial Institute 1640 (RPMI-1640), fetal bovine serum (FBS),l -glutamine and penicillin-streptomycin solution were purchased from Hyclone Laboratories (Logan, UT, USA). Ammonium-chloride-potassium (ACK) buffer used in the process of isolating bone marrow derived dendritic cells from mouse was purchased from Lonza (Allendale, NJ, USA). Recombinant murine granulocyte-macrophage colony stimulating factor (rmGM-CSF) was purchased from Peprotech (Rocky Hill, NJ, USA). Lipopolysaccharide (LPS) from Salmonella enterica serotype enteritidis was purchased from Sigma-Aldrich.
Cell culture media components including Dulbecco’s modified Eagle’s medium (DMEM), Roswell Park Memorial Institute 1640 (RPMI-1640), fetal bovine serum (FBS),
5
Generation of Murine Myeloid-Derived Suppressor Cells
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MDSCs were generated from BM of naïve wt or EGFP-LysM-Tg BALB/c mice. Femurs and tibias were collected under aseptic condition, and BM was flushed out with sterile PBS. After red blood cell lysis, BM cells were counted (the number of cells was usually 3–4×107 per mouse), and seeded in Petri dishes at a density of 5×105 cells per ml of Dulbecco’s Modified Eagle Medium (DMEM; Sigma-Aldrich) containing 10% fetal bovine serum (FBS) (Hyclone, Logan, UT). In preliminary dose-finding experiments the BM cells were cultured for 3 to 7 days in the presence of varying doses of recombinant murine granulocyte macrophage colony stimulating factor (rmGM-CSF; Peprotech, Rocky Hill, NJ) and recombinant murine interleukin-6 (rmIL-6; Peprotech), or with a combination of rmGM-CSF, rmIL-6, and recombinant murine granulocyte colony stimulating factor (rmG-CSF; Peprotech). On the basis of phenotypic and functional characteristics, the optimal protocol for BM-MDSC generation was found to be a 3-day culture of BM cells in the presence of rmGM-CSF, rmIL-6, and rmG-CSF (10 ng/ml each).
DCs, as Ag-presenting cells (APCs), were also generated from BM of naïve wt BALB/c mice by culturing BM cells for 9 days in the presence of 40 ng/ml rmGM-CSF, as described previously [21] (link), [31] (link).
DCs, as Ag-presenting cells (APCs), were also generated from BM of naïve wt BALB/c mice by culturing BM cells for 9 days in the presence of 40 ng/ml rmGM-CSF, as described previously [21] (link), [31] (link).
6
Generation of Murine Dendritic Cells
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DCs were generated as previously described by Lutz et al (40 (link)). Briefly, the bone marrow was flushed from femurs and tibias obtained from 60 female BALB/c mice (6–8 weeks old, 16–18 g). Mice were euthanized by carbon dioxide asphyxiation for approximately 6 min (air displacement rate: 20%/min; carbon dioxide flow rate: 1.7 l/min; the mortality was ensured by cervical dislocation). Cells (1×106 cells/well) were washed twice with PBS and seeded in each well of a 6-well plate in 2 ml RPMI 1640 medium supplemented with 10 ng/ml recombinant murine granulocyte-macrophage colony stimulating factor (rmGM-CSF), 20 ng/ml recombinant murine interleukin (rmIL)-4 (both from PeproTech, Inc.) and 10% FBS at 37°C with 5% CO2 for 8 days. The morphology of DCs was observed and images were captured using an inverted light microscope (Olympus Corporation) at a magnification of ×200.
7
Isolation and Activation of Bone Marrow-Derived Dendritic Cells
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Bone marrow cells were isolated from the femur and tibia of eight-week-old C57BL/6N mice. The isolated bone marrow cells were cultured in RPMI 1640 (Welgene) supplemented with 10% heat-inactivated fetal bovine serum (FBS; HyClone Laboratories, Inc., Logan, UT, USA), 1% penicillin/streptomycin (Gibco, Waltham, MA, USA), 20 ng/ml recombinant murine granulocyte-macrophage colony-stimulating factor (rmGM-CSF; Peprotech, Rocky Hill, NJ, USA), and 55 μM β-mercaptoethanol (Gibco) for 7 days (37°C, 5% CO2). On Day 3, fresh medium was added. On Day 7, floating cells were harvested and labeled with biotin anti-CD11c antibody (BioLegend, San Diego, CA, USA) and subsequently incubated with anti-biotin microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). The CD11c+ cells were isolated using an MS column (Miltenyi Biotec) according to the manufacturer’s instructions. The isolated CD11c+ BMDCs were stimulated with 10 μg/ml periodontal pathogen OMVs in RPMI 1640 complete medium (supplemented with 10% heat-inactivated FBS and 1% penicillin/streptomycin) for 24 h, and the expression of cell surface markers and cytokines was analyzed via flow cytometry and ELISA, respectively.
8
Isolation and Characterization of Murine Bone Marrow-Derived Dendritic Cells
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DCs were isolated from mouse bone marrow as previously described 10 (link), 20 (link). Bone marrow mononuclear cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 20 ng/ml recombinant murine granulocyte-macrophage colony-stimulating factor (rmGM-CSF), and 20 ng/ml recombinant murine interleukin (rmIL)-4 (both from Peprotech, Inc) for 7 days. The images of immature bone marrow-derived DCs (BMDCs) were captured using an inverted light microscope (Olympus Corporation). Immature BMDCs were incubated with CD11c MicroBeads UltraPure (Miltenyi Biotec, Inc.) for 20 min at 4°C following the manufacturer's instructions. The positive fraction containing CD11c+ cells was analyzed by flow cytometry.
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