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Gm csf

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GM-CSF is a cytokine that stimulates the growth and differentiation of hematopoietic precursor cells from various lineages, including granulocytes and macrophages. It is used in laboratory research to study the biology and function of these cell types.

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Ifn γ, by BD (7 mentions) Interleukin 6 (il 6), by BD (6 mentions) Interleukin 4 (il 4), by BD (5 mentions) Tnf α, by BD (4 mentions) Il 10, by BD (3 mentions) Interleukin 4 (il 4), by Thermo Fisher Scientific (3 mentions) Hepes, by Thermo Fisher Scientific (3 mentions) Rpmi 1640, by Thermo Fisher Scientific (3 mentions) Penicillin, by BD (2 mentions) Live dead discriminator, by Thermo Fisher Scientific (2 mentions) Cd56 hcd56 from, by BioLegend (2 mentions) Cd10 hi10a from, by BioLegend (2 mentions) Upr1 kir2dl5, by BioLegend (2 mentions) 181703 kir2dl4, by R&D Systems (2 mentions) Fes172 kir2ds4 and eb6 kir2dl1 s1, by Beckman Coulter (2 mentions) Chl kir2dl2 3 s2, by BD (2 mentions) Dx9 kir3dl1, by BioLegend (2 mentions) Nkvfs1 kir2dl1 2 3 s1 2 4, by Abcam (2 mentions) 5.133 kir3dl2, by Merck Group (2 mentions) Ki647, by BD (2 mentions)

Close spelling variants of this product

Murine GM-CSF (5 citations) GM-CSF-PE (4 citations) Recombinant mouse GM-CSF (3 citations) Human GM-CSF (3 citations) Rat anti-GM-CSF antibody (2 citations) BD OptEIA™ Human GM-CSF ELISA Set (2 citations) GM-CSF (clone MP1–22E9 - (2 citations) Anti-GM-CSF, (2 citations) Recombinant human GM-CSF (2 citations) Anti-GM-CSF PE (2 citations)

45 protocols using gm csf

1

Quantifying GM-CSF Neutralizing Activity

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Neutralizing activity of plasma against GM-CSF was performed by assessing STAT5 phosphorylation in cells stimulated with GM-CSF in the presence of plasma from patients or controls. Briefly, PBMCs (1 × 106 cells/ml) were cultured in complete RPMI 1640 with 10% FCS with plasma from normal subjects or patients and left unstimulated or stimulated with recombinant human GM-CSF (Peprotech) for 30 min at 37 °C. Monocytes were identified by CD14 (BD Pharmingen) surface staining before being fixed and permeabilized for intracellular staining with p-STAT5 (Y694) Ab (BD Bioscience), as described previously [3 ].
In order to approximate the IC50 (concentration of antibodies responsible for 50% of inhibition of GM-CSF-induced STAT5 phosphorylation), the patient plasmas were used at different dilutions (diluted in AB serum from 0.01 to 30%) with a constant concentration of GM-CSF for stimulation (10 ng/ml).
In order to assess the EC50 (concentration of GM-CSF responsible for 50% of STAT5 phosphorylation in the presence of antibodies), the volume of plasma was chosen to get a constant final concentration of antibodies in each tube at 10 MMAU (completed with AB serum); stimulation with GM-CSF varied from 10–1 to 104 ng/ml.
Data were collected using a BD LSR Fortessa (BD Biosciences), analyzed using FlowJo software (TreeStar), and graphed with Prism8 (GraphPad).
Salvator H., Cheng A., Rosen L.B., Williamson P.R., Bennett J.E., Kashyap A., Ding L., Kwon-Chung K.J., Namkoong H., Zerbe C.S, & Holland S.M. (2022). Neutralizing GM-CSF autoantibodies in pulmonary alveolar proteinosis, cryptococcal meningitis and severe nocardiosis. Respiratory Research, 23, 280.
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2

Detailed Protocol for Mouse Bone Marrow-Derived Dendritic Cell Culture and Stimulation

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Mouse bone marrow-derived DCs were generated as previously described [10 (link)]. Briefly, bone marrow precursors were cultured in complete IMDM (10% FBS, antibiotics and 2-mercaptoethanol) containing 3.3 ng/ml GM-CSF (BD Biosciences, San Jose, USA) or 1% conditioned media from GM-CSF-secreting B7H1 cells. At day 6–8, cDCs were stimulated with LPS (100 ng/ml), R848 (1 μg/ml), or CpG 1826 (10 μg/ml) and harvested after 1.5 hours for Western Blot and 6 hours for RT-PCR, while supernatants and cells were collected after 24 hours for ELISA and Flow analysis. Anti-IFNAR (10 μg/ml, clone MAR1–5A3, Leinco Technologies I-400 or Gentex GTX14637) or isotype control antibody (10 μg/ml, IgG1, Leinco Technologies I-102 or Gentex GTX35014) were added 24 hours and again 30 minutes before or 1 hour before TLR stimulation. ERK inhibitor (10 μM, PD98059, Cell Signaling #9900) was used 30 minutes before TLR stimulation. 1μg/ml anti-IL-27 (Polyclonal goat IgG, R&D AF1834) or 1μg/ml IgG control antibodies (R&D AB-108-C) were added one hour prior to TLR stimulation.
Lee M.H., Gallo P.M., Hooper K.M., Corradetti C., Ganea D., Caricchio R, & Gallucci S. (2019). The cytokine network Type I Interferon, IL-27 and IL-10 is augmented in murine and human lupus.. Journal of leukocyte biology, 106(4), 967-975.
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3

Cytokine Profiling by ELISA

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The concentrations of IL-6, IL-8, GM-CSF, G-CSF, TNF-α, and CCL2 in cell supernatant were measured with a sandwich enzyme-linked immunosorbent assay (ELISA) using OptEIA Set human IL-6, IL-8, GM-CSF, TNF-α, and CCL2 (BD Biosciences, San Diego, CA, USA) and G-CSF (Abfrontier, Seoul, Korea) according to the manufacturer's instructions.
Kim E.H., Lee J.S., Lee N.R., Baek S.Y., Kim E.J., Lee S.J, & Kim I.S. (2014). Regulation of Constitutive Neutrophil Apoptosis Due to House Dust Mite Allergen in Normal and Allergic Rhinitis Subjects. PLoS ONE, 9(9), e105814.
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4

Rhinovirus-Induced Maturation of Monocyte-Derived DCs

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Peripheral blood mononuclear cells (PBMCs) were isolated through Ficoll-Hypaque (Sigma, St Louis, MO) density centrifugation. CD14+ cells were enriched (>95%) from PBMCs using positive magnetic affinity column purification (Miltenyi Biotec, Auburn, CA). Cells were suspended in STEM PRO-34 medium (Invitrogen, Carlsbad, CA) supplemented with GM-CSF (1000 U/ml; BD Pharmingen, San Diego, CA), IL-4 (1000 U/ml; BD Pharmingen), penicillin (10,000 U/ml), streptomycin (10 μg/ml) and 10% autologous serum and maintained at 37° C in 5% CO2. Cells were differentiated for 5 days before analysis, with medium and supplements changed at day 3. RV-induced maturation of monocyte-derived DCs was evaluated after the application of ∼350 TCID50 of RV39. After an additional 48 hrs, cells were collected for surface expression of maturation markers. In addition, supernatants and mRNA transcripts were isolated and analyzed for expression of cytokines relevant to DC maturation status and anti-viral immunity or involved in T lymphocyte development and immune deviation.
Steinke J.W., Liu L., Turner R.B., Braciale T.J, & Borish L. (2015). Immune Surveillance by Rhinovirus-Specific Circulating CD4+ and CD8+ T Lymphocytes. PLoS ONE, 10(1), e0115271.
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5

Comprehensive NK Cell Phenotyping

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dNK cells were gated on as live, CD9+CD56+ cells. pbNK cells were gated on as live CD56+CD3− cells. The following Abs were used: Live/Dead discriminator (Life Technologies), CD9 (SN4 or M-L13 from eBioscience or BD Biosciences, respectively), CD56 (HCD56 from BioLegend), and CD3 (SK7) from BD Biosciences. Fibroblasts and macrophages were identified using CD10 (HI10a from BioLegend) and CD14 Abs (MφP9 and HCD14 from BD Pharmingen and BioLegend), respectively. The following Abs were used to stain KIRs: UPR1 (KIR2DL5) from BioLegend and Carlos Vilches (33 (link)); 179315 (KIR2DS4), 143211 (KIR2DL1), and 181703 (KIR2DL4) from R&D Systems; FES172 (KIR2DS4) and EB6 (KIR2DL1/S1) from Beckman Coulter; CHL (KIR2DL2/3/S2) from BD Pharmingen; DX9 (KIR3DL1) from BioLegend; NKVFS1 (KIR2DL1/2/3/S1/2/4) from Abcam; 5.133 (KIR3DL2) from Marco Colonna (34 (link)); and FLAG Abs from Sigma-Aldrich. Intracellular staining was performed according to the manufacturers’ instructions with Abs against Ki647 (BD Pharmingen), CCL3 (R&D Systems), and GM-CSF (BD Biosciences).
Kennedy P.R., Chazara O., Gardner L., Ivarsson M.A., Farrell L.E., Xiong S., Hiby S.E., Colucci F., Sharkey A.M, & Moffett A. (2016). Activating KIR2DS4 Is Expressed by Uterine NK Cells and Contributes to Successful Pregnancy. The Journal of Immunology Author Choice, 197(11), 4292-4300.
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6

Eosinophil/Basophil Progenitor Assay

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Enriched CD34+ progenitors (8000 cells/well) were cultured in duplicates in 0.9% methylcellulose (Sigma Aldrich, St. Louis, MO, USA) with Iscove's 2+ (modified Dulbecco's medium (Gibco, Burlington, Ontario, Canada) supplemented with FBS, penicillin–streptomycin, and 2-ME) and IL-3 (1 ng/mL), IL-5 (1 ng/mL), or GM-CSF (10 ng/mL; BD Biosciences, Mississauga, ON, Canada) in the presence or absence of TSLP (10 ng/mL; PeproTech, Rocky Hill, NJ, USA) in 12-well plates (Corning Costar, Corning, NY, USA). In some experiments, cells were treated with anti-TSLP (Amgen, Seattle, WA, USA), anti-TSLPR (R&D Systems, Minneapolis, MN, USA), anti-TNFα (R&D), or isotype control (each at 10 µg/mL). Treatment with the indicated stimulatory/inhibitory conditions had no effects on cell viability as determined by trypan blue exclusion. Cultures were incubated for 14 days (37°C, 5% CO2). Eo/B CFU were enumerated using inverted light microscopy (colonies were defined as tight, granular clusters ≥40 cells).
Hui C.C., Rusta-Sallehy S., Asher I., Heroux D, & Denburg J.A. (2014). The effects of thymic stromal lymphopoietin and IL-3 on human eosinophil–basophil lineage commitment: Relevance to atopic sensitization. Immunity, Inflammation and Disease, 2(1), 44-55.
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7

Bone Marrow-Derived Dendritic Cell Activation

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Bone marrow cells were isolated from the tibias and femurs of B6 mice, cultured in vitro in RPMI-1640 with 10% FCS, 2 mM L-glutamine, 30 mM HEPES, 0.05 mM β-mercaptoethanol and 0.05 mg ml−1 gentamycin, supplemented with 1,000 U ml−1 GM-CSF (BD Biosciences) and 100 U ml−1 IL-4 (eBioscience). Medium was changed on day 2 and the top half of the medium was changed on day 5 of culture. On day 7, BMDCs were harvested, enriched for CD11c+ cells (Miltenyi Biotec) and activated overnight with NIH 3T3 cells expressing CD40L70 (link) (provided by R. Lapoint, University of Montreal). Activated BMDCs were pulsed with 50 μg ml−1 of β-gal721-739 (AENLSVTLPAASHAIPHLT, GenScript) for 3 h, washed and 1 × 105 BMDCs were injected IV.
Rouhani S.J., Eccles J.D., Riccardi P., Peske J.D., Tewalt E.F., Cohen J.N., Liblau R., Mäkinen T, & Engelhard V.H. (2015). Roles of lymphatic endothelial cells expressing peripheral tissue antigens in CD4 T-cell tolerance induction. Nature Communications, 6, 6771.
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8

SKNO-1 Cell Culture Protocol

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SKNO-1 cells were purchased from DSMZ (Braunschweig, Germany) and cultured in RPMI 1640 with 20% FCS, 1% penicillin-streptomycin, and 10 ng ml-1 GM-CSF (BD Biosciences).
Micol J.B., Pastore A., Inoue D., Duployez N., Kim E., Lee S.C., Durham B.H., Chung Y.R., Cho H., Zhang X.J., Yoshimi A., Krivtsov A., Koche R., Solary E., Sinha A., Preudhomme C, & Abdel-Wahab O. (2017). ASXL2 is essential for haematopoiesis and acts as a haploinsufficient tumour suppressor in leukemia. Nature Communications, 8, 15429.
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9

Quantifying Cytokine Secretion in RevCAR T Cell Assays

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Triplets of 5*103 tumor cells were seeded in a 96-well cell culture plate and cultivated with RevCAR T cells in the presence or absence of RevTMs. Supernatants of co-culture assays were harvested after 24 or 48 h, as indicated in the figure legends. Analysis of secreted cytokines was performed using the OptEIA Human IFN-γ (Cat#555142), GM-CSF (Cat#555126), IL-2 (Cat#555190), and TNF (Cat#555212) ELISA Sets purchased from BD BioSciences or the human MACSPlex Cytokine 12 Kit (Miltenyi Biotec #130-099-169) as described in the manufacturer’s manuals.
Feldmann A., Hoffmann A., Bergmann R., Koristka S., Berndt N., Arndt C., Rodrigues Loureiro L., Kittel-Boselli E., Mitwasi N., Kegler A., Lamprecht C., González Soto K.E, & Bachmann M. (2020). Versatile chimeric antigen receptor platform for controllable and combinatorial T cell therapy. Oncoimmunology, 9(1), 1785608.
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10

Isolation and culture of murine neutrophils and dendritic cells

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Protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at the National University of Singapore. Bone marrow derived neutrophils and DC were generated as previously described [16 (link)]. In brief, neutrophils were derived by positive selection with anti-Ly6G microbead kit (Miltenyi Biotec, Germany) and were at least 95% positive for Ly6G, by flow cytometry. DCs were obtained from the bone marrow derived cells after 9 days of culture (with fresh media replacement every other day) in RPMI 1640 supplemented with 10% heat-inactivated FCS, 50 μM 2-mercaptoethanol, 1% penicillin, streptomycin, glutamine, MEM (minimum essential medium), and 0.1% sodium pyruvate with 40 ng/mL of GM-CSF (BD Bioscience, USA). The DCs were at least 95% positive for CD11c, by flow cytometry. The media used for both neutrophil and DC experiments were DMEM supplemented with 10% fetal bovine serum (Hyclone, USA), 2 mM L-glutamine (Gibco, Japan), 50 μg/mL penicillin G (Sigma Aldrich, USA), and 50 μM of 2-mercaptoethanol (Merck, Germany) with the addition of 20 ng/mL of GM-CSF for the culture of dendritic cells.
T lymphocytes were isolated from spleens of naive C57BL/6 mice and enriched with the EasySepTM T cell isolation kit (STEMCELL Technologies, Vancouver, Canada). The desired fraction was about 95–98% CD3 positive.
Cai S., Kandasamy M., Rahmat J.N., Tham S.M., Bay B.H., Lee Y.K, & Mahendran R. (2016). Lactobacillus rhamnosus GG Activation of Dendritic Cells and Neutrophils Depends on the Dose and Time of Exposure. Journal of Immunology Research, 2016, 7402760.
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