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Granulocyte macrophage colony stimulating factor

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Granulocyte-macrophage colony-stimulating factor is a cytokine that stimulates the production and differentiation of granulocytes and monocytes from bone marrow progenitor cells.

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Lab products found in correlation

Penicillin, by Lonza (2 mentions) L glutamine, by Lonza (2 mentions) Dulbecco s modified eagle s medium, by Lonza (2 mentions) Fetal bovine serum (fbs), by Lonza (2 mentions) Pe labeled anti mouse cd11c, by BioLegend (1 mentions) Heat inactivated fetal bovine serum, by Lonza (1 mentions) Streptomycin, by Lonza (1 mentions) Human cd14 microbeads, by Miltenyi Biotec (1 mentions) Rpmi 1640 medium, by Lonza (1 mentions) Sodium pyruvate, by GE Healthcare (1 mentions) Non essential amino acids (neaa), by GE Healthcare (1 mentions) Dynabeads untouched human t cells kit, by Thermo Fisher Scientific (1 mentions) Gentamicin sulfate, by Lonza (1 mentions) Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (1 mentions) Il 15, by Immunotools (1 mentions) Prostaglandin e2, by Pfizer (1 mentions) Interferon γ, by Immunotools (1 mentions) Penicillin and streptomycin, by Lonza (1 mentions) E coli lps, by Merck Group (1 mentions) Cd14 microbead, by Miltenyi Biotec (1 mentions)

7 protocols using granulocyte macrophage colony stimulating factor

1

Generation of mouse bone marrow-derived dendritic cells

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Bone marrow cells were isolated from the tibia of C57BL/6
mice and
cultured in Dulbecco’s modified Eagle’s medium (Lonza,
Basel, Switzerland) supplemented with 10% heat-inactivated fetal bovine
serum (Lonza), 2 mM l-glutamine (Lonza), 100 U/mL penicillin
(Lonza), 100 μg/mL streptomycin (Lonza), and 20 ng/mL granulocyte-macrophage
colony-stimulating factor (ImmunoTools, Friesoythe, Germany) for 7
days at 37 °C and 5% CO2. The purity of the BMDCs
was evaluated with PE-labeled anti-mouse CD11c (Biolegend, San Diego,
CA, U.S.A.) by flow cytometry with >90% shown to be CD11c positive.
Guzelj S., Nabergoj S., Gobec M., Pajk S., Klančič V., Slütter B., Frkanec R., Štimac A., Šket P., Plavec J., Mlinarič-Raščan I, & Jakopin Ž. (2021). Structural Fine-Tuning of Desmuramylpeptide NOD2 Agonists Defines Their In Vivo Adjuvant Activity. Journal of Medicinal Chemistry, 64(11), 7809-7838.
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2

Isolation and Differentiation of Human Monocyte-Derived Dendritic Cells

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Human erythrocyte- and plasma depleted blood were obtained from healthy volunteers from Ostfold Hospital Trust, Fredrikstad, Norway in accordance with institutional ethical guidelines and with approval from the Regional Committee of Medical and Health Research Ethics with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation on a Lymphoprep gradient (Fresenius Kabi). Human T cells were isolated from PBMCs by negative selection using Dynabeads Untouched Human T Cells Kit (Thermo Fisher). CD14+ cells were isolated by positive selection using human CD14 MicroBeads (Miltenyi Biotech). To develop immature monocyte-derived dendritic cells (MoDCs) CD14+ cells were cultivated for 6 days in RPMI 1640 medium supplemented with 10% heat inactivated fetal calf serum and 25 μg/ml gentamicin sulfate (Lonza), 1 mM sodium pyruvate and 100 μM non-essential amino acids (both from PAA Laboratories), 25 ng/ml interleukin 4 and 50 ng/ml granulocyte macrophage colony stimulating factor (both from ImmunoTools).
Indrelid S., Kleiveland C., Holst R., Jacobsen M, & Lea T. (2017). The Soil Bacterium Methylococcus capsulatus Bath Interacts with Human Dendritic Cells to Modulate Immune Function. Frontiers in Microbiology, 8, 320.
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3

Cytokine Profiling of Stimulated Monocyte-Derived Dendritic Cells

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Preparation of monocyte‐derived dendritic cells was performed by harvesting MNCs as described above and subsequent isolation of CD14+ monocytes by magnetic sorting (MojoSort, BioLegend). MoDCs were generated by addition of interleukin 4 and granulocyte‐macrophage colony stimulating factor (both 500 U/mL, ImmunoTools, Germany) to monocytes in 6‐well plates for 7 days as described previously, harvested, and resuspended in complete RPMI for stimulation experiments. MoDCs were grown and incubated in a humidified atmosphere of 5 % carbon dioxide at 37 °C. For the stimulation, compounds were added to moDCs (1x106/mL) in the indicated concentrations and incubated for 20 h. Cytokine releases of stimulated moDCs were quantified after 20 h by using commercial ELISA Kits (Life Technologies GmbH, Darmstadt, Germany) specific for IL‐6, IL‐10 and IL‐12‐p70 in supernatants. Data were combined from n=3 independent experiments; error bars indicate standard error of the mean.
Strobl S., Hofbauer K., Heine H, & Zamyatina A. (2022). Lipid A Mimetics Based on Unnatural Disaccharide Scaffold as Potent TLR4 Agonists for Prospective Immunotherapeutics and Adjuvants. Chemistry (Weinheim an Der Bergstrasse, Germany), 28(35), e202200547.
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4

Rapid Generation of IL-15 Dendritic Cells

Cited 2 times
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IL-15 DCs were prepared as per our previously reported rapid DC culture protocol [11 (link), 12 , 14 (link)]. Briefly, monocytes were seeded in Roswell Park Memorial Institute medium (RPMI; Life Technologies) supplemented with 2.5% heat-inactivated human AB serum (hAB; Invitrogen, Merelbeke, Belgium) at a final concentration of 1.0–1.2 × 106 cells/mL. Differentiation was induced with 800 IU/mL granulocyte macrophage colony-stimulating factor and 200 ng/mL IL-15 (Immunotools, Friesoythe, Germany). A TLR-activating maturation cocktail, comprising R848 (3 μg/mL; Alexis Biochemicals, San Diego, USA), tumor necrosis factor-α (2.5 ng/mL), interferon-γ (250 ng/mL; Immunotools) and prostaglandin E2 (1 μg/mL; Pfizer, Puurs, Belgium), was added after 24–48 hours of differentiation for 18–20 hours. All components were bought from Invitrogen, unless stated otherwise. Control 7-day IL-4 DCs from the same blood donors were prepared as previously described in detail [11 (link)]. All subsequent experiments were performed using the obtained activated IL-15 DCs and IL-4 DCs. For the collection of x-hour wash-out supernatant, mature DCs were harvested, washed thoroughly and resuspended in fresh medium, RPMI + 2.5% hAB, at a concentration of 1 × 106 cells/mL. After x hours of culturing in low absorbing polypropylene tubes, cell-free supernatant was collected and frozen at −20°C until further use.
Van Acker H.H., Beretta O., Anguille S., Caluwé L.D., Papagna A., Van den Bergh J.M., Willemen Y., Goossens H., Berneman Z.N., Van Tendeloo V.F., Smits E.L., Foti M, & Lion E. (2017). Desirable cytolytic immune effector cell recruitment by interleukin-15 dendritic cells. Oncotarget, 8(8), 13652-13665.
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5

Isolation and Differentiation of Monocyte-Derived Dendritic Cells

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Human monocytes were isolated from buffy coats of healthy donors (Sanquin, Amsterdam, The Netherlands) or from fresh blood of healthy volunteers. All blood donors gave informed consent. Monocytes were isolated from peripheral blood mononuclear cells using CD14 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) as previously described37 (link) and differentiated into immature DCs in RPMI in the presence of 10% fetal calf serum, 10.000 Uml − 1 penicillin and streptomycin (BioWhittaker, Walkersville, MD, USA), 10.000 Uml − 1 glutamine (Invitrogen, Thermo Scientific, Waltham, MA, USA) and 250 IU ml− 1 IL-4 and 120 IU ml− 1 granulocyte-macrophage colony-stimulating factor (Immunotools, Friesoythe, Germany). For all experiments, monocyte-derived DCs were used after 4 days of differentiation, and E. coli LPS (Sigma, St Louis, MO, USA) was used at a final concentration of 10 ng ml− 1. All experiments were performed at least three times, in separate experiments using DCs derived from different donors.
Klaver E., van der Pouw Kraan T., Laan L., Kringel H., Cummings R., Bouma G., Kraal G, & van Die I. (2015). Trichuris suis soluble products induce Rab7b expression and limit TLR4 responses in human dendritic cells. Genes and immunity, 16(6), 378-387.
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6

Isolation and Differentiation of Human Monocyte-Derived Dendritic Cells

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Monocyte isolation was performed as described.25 (link) Briefly, peripheral blood mononuclear cells were collected from centrifuged buffy coat and incubated with RosetteSep human monocyte enrichment kit (StemCell Technologies SARL, Grenoble, France), according to manufacturer’s instructions. The mixture was diluted with phosphate buffered saline (PBS) supplemented with heat-inactivated fetal bovine serum (FBS) (Lonza, Basel, Switzerland), layered over Histopaque®-1077 (Sigma Aldrich GmbH, Steinheim, Germany), and centrifuged as before. The enriched monocyte layer was collected and washed with PBS. Population purity was evaluated and over 70% of the cells were found to be CD14 positive.25 (link) Cells were plated at 1×106 cells/mL in complete culture media (Roswell Park Memorial Institute-1640+Glutamax, with 10% heat-inactivated FBS and 1% penicillin G-streptomycin [Invitrogen, Paisley, UK]), further supplemented with 50 ng/mL interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor (Immunotools, Friesoythe, Germany). Monocyte-derived DCs were differentiated for 4–5 days to obtain immature DCs.26 (link),27 (link)
Barbosa J.P., Neves A.R., Silva A.M., Barbosa M.A., Reis M.S, & Santos S.G. (2016). Nanostructured lipid carriers loaded with resveratrol modulate human dendritic cells. International Journal of Nanomedicine, 11, 3501-3516.
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7

Isolation and Culture of Bone-Marrow Derived Dendritic Cells

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Bone-marrow cells
were isolated from the tibia of C57BL/6 mice and cultured in Dulbecco’s
modified Eagle’s medium (Lonza, Basel, Switzerland) supplemented
with 10% heat-inactivated fetal bovine serum (Lonza), 2 mM l-glutamine (Lonza), 100 U/mL penicillin (Lonza), 100 μg/mL
streptomycin (Lonza), and 20 ng/mL granulocyte-macrophage colony-stimulating
factor (ImmunoTools, Friesoythe, Germany) for 7 days at 37 °C
and 5% CO2. The purity of the BMDCs was evaluated with
a PE-labeled anti-mouse CD11c antibody (Biolegend, San Diego, CA)
by flow cytometry, with > 90% shown to be CD11c-positive.
Guzelj S., Weiss M., Slütter B., Frkanec R, & Jakopin Ž. (2022). Covalently Conjugated NOD2/TLR7 Agonists Are Potent and Versatile Immune Potentiators. Journal of Medicinal Chemistry, 65(22), 15085-15101.
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