Human and canine DCs were generated using a three-step protocol as described previously [23 (link)]. First, peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using standard procedures (Ficoll-Paque Plus®; GE Healthcare, Uppsala, Sweden). Second, monocytes were purified from PBMCs by magnetic cell sorting using an anti-CD14 monoclonal antibody conjugated to magnetic beads (MACS®; Miltenyi Biotec, Bergisch Gladbach, Germany). Third, DCs were generated by culturing CD14+ monocytes at 1 × 106 cells/mL for 6 days in RPMI-1640 (Life Technologies, Gaithersburg, MD, USA), supplemented with 10% foetal bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin, 2 mM l -glutamine (Gibco Invitrogen Corp., Grand Island, NY, USA), 100 U/mL polymixin B (Sigma-Aldrich, St. Louis, MO, USA), and specific recombinant human (rh) or canine (rc) differentiation factors. In particular, canine monocyte cultures were supplemented with 40 ng/mL rhGM-CSF and 30 ng/mL rcIL-4 (R&D Systems Inc., Minneapolis, MN, USA), while human monocyte cultures were supplemented with 20 ng/mL rhGM-CSF and rhIL-4 (R&D Systems Inc.) every 2 days.
Rhgm csf
Manufactured by R&D Systems
Sourced in United States, Mongolia, United Kingdom
RhGM-CSF is a recombinant human Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) protein. GM-CSF is a cytokine that regulates the production, differentiation, and function of granulocytes and macrophages.
Automatically generated - may contain errors
Lab products found in correlation
Rhil 4, by R&D Systems (23 mentions)
Rhil 3, by R&D Systems (7 mentions)
2 mercaptoethanol, by Merck Group (7 mentions)
Methylcellulose, by STEMCELL (6 mentions)
Trypan blue, by Thermo Fisher Scientific (6 mentions)
Rhepo, by R&D Systems (6 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (6 mentions)
Bovine serum albumin (bsa), by Merck Group (6 mentions)
Ficoll paque plus, by GE Healthcare (4 mentions)
Rhm csf, by R&D Systems (4 mentions)
Magnetic activated cell sorting (macs), by Miltenyi Biotec (4 mentions)
Tnf α, by R&D Systems (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
L glutamine, by Thermo Fisher Scientific (3 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Magnetic microbead, by Miltenyi Biotec (2 mentions)
Fetal calf serum (fcs), by Merck Group (2 mentions)
Rhil 8, by R&D Systems (2 mentions)
Interleukin 4 (il 4), by R&D Systems (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
49 protocols using rhgm csf
1
Generation of Human and Canine Dendritic Cells
2
Differentiation of Monocyte-Derived Dendritic Cells
3
Isolation and Maturation of Dendritic Cells
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Peripheral blood mononuclear cells (PBMCs) were obtained from blood cells collected at the Etablissement Français du Sang (EFS, Rungis, France) as buffy coat preparations from anonymous healthy donors who gave informed consent, in accordance with EFS guidelines. PBMCs were isolated by Ficoll-Paque PLUS density gradient centrifugation (GE Healthcare, Buc, France). Monocyte-derived DCs were generated from plastic-adherent PBMCs after 4 or 5 days of culture in AIM-V medium (Invitrogen, Villebon-sur-Yvette, France) supplemented with 1,000 U/mL of recombinant human IL-4 (rh-IL-4; R&D systems, Lille, France) and of rh-GM-CSF (R&D systems). Monocyte-derived DCs were matured with 1 μg/mL of lipopolysaccharide (LPS, Sigma, St. Louis, MO, USA) for 2 days at 37°C. CD4 T cells were isolated from autologous PBMCs by positive selection using an anti-CD4 monoclonal antibody coupled to magnetic microbeads (Miltenyi Biotech, Paris, France) and magnetic cell sorting, as recommended by the manufacturer. The HLA-DR genotypes were determined using the Gold SSP DRB1 typing kit (Invitrogen) after DNA extraction from PBMCs with the NucleoSpin Blood L Kit (Macherey Nagel, Hoerdt, France).
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Allergic Responses Modulation Protocols
5
Inducing MDSC Death with CD33 Antibody
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Example 48
Human peripheral blood cells were ficolled, red blood cells were lysed with ACK lysis buffer, and cells were washed 3 times before plating in RPMI with 10% FBS, Pen/Strep, 1 mM glutamine, non-essential amino acids, and sodium pyruvate. Human peripheral blood mononuclear cells (PBMCs) were cultured for one week at 37° C. in 5% CO2, with the following cytokines: 10 ng/ml rhGM-CSF (R&D), 10 ng/ml rhIL-8 (R&D), and 10 ng/ml rhIL-6 (Sigma). The mIgG1 (BioXcell) isotype, or the CD33 antibody 2F5 or 6C7 was then added to flasks. Every 2-3 days, media, cytokines, and antibodies (where applicable) were replenished. After 7 days of culture, cells were harvested by scraping and triterating, and resuspended in PBS with 2% FBS and 1 mM EDTA for FACS analysis. For assessment of live and dead cell populations, Live/Dead Fixable Aqua dead cell stain (Thermo Fisher) was diluted 1:500 in PBS with 2% FBS and 1 mM EDTA and incubated for 30 minutes on ice. Cell were washed 3 times in buffer and analyzed on a BD FACS Canto (BD Biosciences) for forward and side scatter and AmCyan fluorescence. Data were analyzed with FlowJo (Tree Star Software).
As shown in
6
Hormonal Regulation of CML Cell Adhesion and Migration
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Quiescent primary CML cells were stimulated with FSH (5 IU/mL), LH (5 IU/mL), prolactin (1 μg/mL), estradiol (100 nM), and progesterone (100 nM). Cells (2.5 × 104/mL) were added to fibronectin-coated 96- well plates and incubated for 5 min at 37°C. Following incubation, the plates were washed and adherent cells were counted.
For cell migration and granulocyte/macrophage colony-forming unit (CFU-GM) assays, primary CML cells were resuspended in assay medium (RPMI medium containing 0.5% BSA). FSH (5 IU/mL), LH (5 IU/mL), estradiol (100 nM), or progesterone (100 nM) were added to the lower chambers of a Transwell plate. Next, cells were loaded onto the upper chambers, and then incubated for 3 h at 37°C and 5% CO2. The migrated CML cells were afterwards harvested, scored, and subjected to CFU- GM colony assay in which the following concentrations of growth factor and cytokine were respectively used: rhGM-CSF (25 ng/mL) and rhIL-3 (10 ng/mL) as previously described (both reagents were purchased from R & D Systems; Minneapolis, MN, USA) [8 (link)].
For cell migration and granulocyte/macrophage colony-forming unit (CFU-GM) assays, primary CML cells were resuspended in assay medium (RPMI medium containing 0.5% BSA). FSH (5 IU/mL), LH (5 IU/mL), estradiol (100 nM), or progesterone (100 nM) were added to the lower chambers of a Transwell plate. Next, cells were loaded onto the upper chambers, and then incubated for 3 h at 37°C and 5% CO2. The migrated CML cells were afterwards harvested, scored, and subjected to CFU- GM colony assay in which the following concentrations of growth factor and cytokine were respectively used: rhGM-CSF (25 ng/mL) and rhIL-3 (10 ng/mL) as previously described (both reagents were purchased from R & D Systems; Minneapolis, MN, USA) [8 (link)].
7
Monocytes Differentiation into M1/M2/IL-17 Macrophages
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The monocytes (4 × 105/ml) were cultured in RPMI 1640 medium supplemented with 2 mM glutamine, antibiotics and 10% fetal calf serum in the presence of 40 ng/ml recombinant human (rh) GM-CSF (rhGM-CSF was donated by Probiomed, México City, México) for M1 or 50 ng/ml of rh macrophage colony-stimulating factor (M-CSF) (R&D Systems, Minneapolis, MN, USA) for M2 for 5 days (the culture medium was replaced every 2 days with fresh culture medium containing rhGM-CSF or rhM-CSF). For complete polarization, M1 were stimulated with 100 ng/ml LPS (Sigma-Aldrich, St. Louis, MO, USA) and 25 ng/ml IFN-γ (R&D Systems, Minneapolis, MN, USA), and M2 were cultured with 10 ng/ml IL-4 and 40 ng/ml IL-13 (R&D Systems, Minneapolis, MN, USA) for 24 h. In contrast, monocytes were cultured with 60 ng/ml rhIL-17 (R&D Systems, Minneapolis, MN, USA) for 6 days (the culture medium was replaced with fresh culture medium containing rhIL-17 every 2 days) to obtain IL-17 differentiated macrophages. For M0 macrophages (M0), monocytes were cultured only in culture medium.
8
Hematopoietic Stem Cell Expansion
9
Isolation and Differentiation of Dendritic Cells from PBMCs
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For each subject, peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using standard procedures (Ficoll-Paque Plus; GE Healthcare, Uppsala, Sweden). For generating a purified population of immature dendritic cells, monocytes were purified from PBMCs by magnetic cell sorting separation (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany). Briefly, PBMCs were incubated with an anti-CD14 monoclonal antibody conjugated to magnetic beads for 15 min at 4°C, loaded onto LS columns and then separated in the magnetic field of a cell separator (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany). The retained CD14+ cells, which correspond to monocytes, were then flushed out and washed twice in phosphate-buffered saline. Monocytes were then immediately differentiated to dendritic cell by culturing at 106 cells/mL in RPMI-1640 medium supplemented with 10% fetal calf serum (Gibco Invitrogen Corp., Grand Island, NY, USA) and 20 ng/mL rhGM-CSF and rhIL-4 (R&D Systems Inc., Minneapolis, MN, USA) for 6 d at 37°C.
10
Hematopoietic Cell Culture Protocol
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