Anti gm csf

Manufactured by R&D Systems

Sourced in United States

Anti-GM-CSF is a laboratory reagent that can be used to detect and quantify granulocyte-macrophage colony-stimulating factor (GM-CSF) in various sample types. It is a neutralizing antibody that binds to GM-CSF, a cytokine that plays a role in the regulation of white blood cell production and function. Anti-GM-CSF can be used in a variety of immunoassay techniques to measure GM-CSF levels.

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GM-CSF

8 protocols using anti gm csf

Investigating T Cell Responses to GM-CSF and CXCL11

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To examine the effects of GM-CSF, CXCL11 and their blockade on T cell responses, human monocytes and naïve T cells were isolated from peripheral blood of healthy volunteers by magnetic cell isolation according to the company’s instructions (Miltenyi Biotec). Purified monocytes at 2 × 10⁴ were seeded to U bottom 96 well plates and cultured in IMDM culture medium, which contained 10% FBS and 1% penicillin-streptomycin, in 7 groups with different treatment conditions including PBS, GM-CSF (10 ng/ml), Anti-GM-CSF (10 µg/ml), RHCXCL11(15 ng/ml) (R&D Systems), RHCXCL11 + GM-CSF, Anti CXCL11 (10 µg/ml) (R&D Systems, USA), and Anti-GM-CSF + Anti-CXCL11 for 24 hours. The following day, isolated naïve T cells were labeled with Cell Trace Violet (Thermo Fisher) following the manufacturer’s instructions. T cells were then stimulated with the soluble anti-CD3 antibody at a concentration of 1 µg/ml. T cells were then added (1 × 10⁵ cells per well) to the same monocyte wells for cell-cell interactions and kept humidified in a 5% CO2 incubator at 37 °C for 72 hours. After that time, cell culture supernatant was carefully removed and immediately frozen at −20 °C. To measure the proliferative capacity of T cells, CFSE intensity in the cells was assessed by flow cytometry.

Lotfi N., Zhang G.X., Esmaeil N, & Rostami A. (2020). Evaluation of the effect of GM-CSF blocking on the phenotype and function of human monocytes. Scientific Reports, 10, 1567.

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Cytokine Regulation of CD4+ T Cell

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CD4⁺CCR6⁺CXCR3^- T cells were separated by cell sorting; CD11C⁺ APCs were separated using a CD11C positive selection kit (STEMCELL). Sorted CD4⁺CCR6⁺ CXCR3^- T cells and CD11C⁺ APCs were co-cultured at a ratio of 2:1 with or without anti-GM-CSF (10 µg/mL; R&D Systems) or IL-38(10 ng/mL) for 24 hours. IL-23 levels in the supernatants were measured using mouse IL-23 ELISA kit (Thermo Fisher Scientific).

Li H., Zhu L., Wang R., Xie L., Chen Y., Duan R., Liu X., Huang Z., Chen B., Li Z., Wang X, & Su W. (2021). Therapeutic Effect of IL-38 on Experimental Autoimmune Uveitis: Reprogrammed Immune Cell Landscape and Reduced Th17 Cell Pathogenicity. Investigative Ophthalmology & Visual Science, 62(15), 31.

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GIFT4 Signaling and B-cell Activation

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GIFT4, GM-CSF, IL-4 and IFN-γ were quantified with ELISA kits (eBiosciences, San Diego, CA). GIFT4 were profiled by Western blot (WB) with anti-GM-CSF or anti-IL-4 antibodies (R&D systems). Murine B-cells (10⁶ cells/ml) treated with GIFT4, GM-CSF and IL-4 with or without JAK inhibitors were lysed with buffer as described (21 (link)). STAT phosphorylation in B-cells was determined by WB with anti-pSTAT1 (Tyr701, D4A7), anti-pSTAT3 (Tyr705, D3A7), anti-pSTAT5 (Tyr694, D47E7), anti-pSTAT6 (Tyr641, C11A12), or anti-STAT antibodies (Cell Signaling). For immunoprecipitation of GMCSFRβ by common γc receptor after GIFT4 or GM-CSF and IL-4 stimulation, the B-cells were lysed with buffer as described (21 (link)). One mg of protein per sample was incubated with 2 mg of anti-common γc or anti-GMCSFR antibodies, or IgG isotype control (Santa Cruz Biotechnology, CA) overnight at 4°C. The sample-antibody complexes were incubated with protein A agarose for 2 hour at 4°C. The bound proteins were eluted for WB with anti-GMCSFR or anti-IL4R antibodies.

Deng J., Yuan S., Pennati A., Murphy J., Wu J.H., Lawson D, & Galipeau J. (2014). Engineered fusokine GIFT4 licenses the ability of B cells to trigger a tumoricidal T cell response. Cancer research, 74(15), 4133-4144.

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Multiparameter Flow Cytometry Panel

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The following antibodies and cell tracer were used staining for flow analysis: FITC-conjugated anti-CD158b (BD Biosciences, San Jose, CA), anti-IFN-γ (eBioscience, San Diego, CA); CFSE; PE-conjugated anti-IFN-γ (BD Biosciences, San Jose, CA), anti-TNF-α, anti-IL-22, anti-granzyme B (eBioscience, San Diego, CA), anti-GM-CSF (R&D Systems, Minneapolis, MN); PerCP-conjugated anti-CD3 (BD Biosciences, San Jose, CA); APC-conjugated anti-CD158a (Miltenyi Biotec, Bergisch Gladbach, Germany), anti-CD4 (BD Biosciences, San Jose, CA), anti-IL-17A (eiBoscience, San Diego, CA); strepavidin-PerCP; PE-Cy7-conjugated anti-CD56, anti-CD14 (BD Biosciences, San Jose, CA), Vioblue-conjugated anti-3DL1 (Miltenyi Biotec, Bergisch Gladbach, Germany), eFluor 650NC-conjugated anti-CD3 (ebBioscience, San Diego, CA). anti-mouse IgG κ/Negative Control Compensation Particles. The use of antibody for staining was performed per manufacturer’s instructions with proper titrations. Antibodies used for cytokine assays are IL-2, IL-6, IL-10, IL-15, IL-13, CCL-4 (MIP-1β), CCL-5, CXCL-10, CCL-2, CXCL-8, IFN-γ, TNF-α, TNF-β, granzyme B, TGF-β1 (R&D Systems, Minneapolis, MN), IL-4, IL-12, GM-CSF, and perforin (eBioscience, San Diego, CA).

Lin L., Ma C., Wei B., Aziz N., Rajalingam R., Yusung S., Erlich H.A., Trachtenberg E.A., Targan S.R., McGovern D.P., Heath J.R, & Braun J. (2014). Human Natural Killer Cells Licensed by KIR Genes Have an Altered Cytokine Program That Modifies CD4+ T Cell Function. Journal of immunology (Baltimore, Md. : 1950), 193(2), 940-949.

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Th17 Cells and APC Interaction

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CD4⁺CCR6⁺CXCR3⁻ T cells (Th17 cells) were isolated by cell sorting and CD11C⁺ APCs were isolated by CD11C positive selection kit (STEMCELL, Vancouver, Canada). Then, sorted CD4⁺CCR6⁺CXCR3⁻ T cells and CD11C⁺ APCs were co-cultured at a ratio of 2:1 with or without anti-GM-CSF (10 g/mL, R&D Systems) for 24 h. The supernatants were used to measure IL-23 by ELISA kit (Invitrogen, Carlsbad, CA, USA).

Li H., Zhu L., Wang R., Xie L., Ren J., Ma S., Zhang W., Liu X., Huang Z., Chen B., Li Z., Feng H., Liu G.H., Wang S., Qu J, & Su W. (2021). Aging weakens Th17 cell pathogenicity and ameliorates experimental autoimmune uveitis in mice. Protein & Cell, 13(6), 422-445.

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Monocyte Viability Assay with Cytokines

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After thawing, CD14+ monocytes were counted and resuspended in αMEM medium supplemented with 20% FBS. In a 96-well plate, we added 50 μL of cells in each well (45,000 cells/well) and then 50 μL of M-CSF, IL-34, and GM-CSF (all from R&D systems) diluted in 2% RPMI to obtain a 25, 50, and 10 ng/mL final concentration, respectively, or 50 μL of RA SF conditioned media. Medium (50 μL; 2% FBS) without any cytokine was used as control condition (CT). After 3 days, cell viability was assessed by WST-1 cell proliferation Reagent (Roche, France) by adding 10 μL per well. The formazan dye obtained from WST-1 cleavage by mitochondrial dehydrogenase was read by a microplate reader Wallac 1420 Victor 2 (Perkin Elmer, USA) between 420 and 480 nm after incubation for 4 hours at 37°C. Antibodies used to identify cytokines implicated in monocyte viability were as follows: anti M-CSF (R&D Systems), anti-IL-34 (Diaclone, INSERM UMR957), and anti-GM-CSF (R&D Systems) at 2, 10, and 5 μg/mL, respectively. We have previously tested different antibody concentrations to determine the most appropriate concentrations.

Darrieutort-Laffite C., Boutet M.A., Chatelais M., Brion R., Blanchard F., Heymann D, & Le Goff B. (2014). IL-1β and TNFα Promote Monocyte Viability through the Induction of GM-CSF Expression by Rheumatoid Arthritis Synovial Fibroblasts. Mediators of Inflammation, 2014, 241840.

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Neutrophil and CD8+ T Cell Migration Assays

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Migration of neutrophils, isolated from PBMC of HDs by magnetic beads (EasyStep enrichment kit, StemCell Technologies), was assessed in transwell plates (5 µm pore size, Corning Costar), towards Th17 supernatants or recombinant cytokines (IL-17, R&D Systems, 50 ng/mL; IL-8, R&D Systems, 100 ng/mL; GM-CSF, R&D Systems, 100 ng/mL), for 90 min at 37°C. In specific experiments, anti-IL-8 or anti-GM-CSF antibodies (10 μg/mL, R&D Systems) were added to Th17 supernatants.
Migration of CD8+ T cells, sorted from PBMC of HDs by magnetic microbeads (Miltenyi Biotec) and pre-activated overnight with anti-CD3/CD28 antibodies, was evaluated in transwell plates (5 µm pore size) towards Th17 supernatants, supernatants of HMEC cells, untreated, or exposed overnight to Th17 supernatants or recombinant proteins (IL-17, 50 ng/mL; TNF-α, 1 ng/mL, R&D Systems; IFN-γ, 1 ng/mL, Biolegend; or their combination), or towards CCL5 (60 and 200 ng/mL, R&D Systems) and/or CCL20 (300 and 1000 ng/mL, R&D Systems). Depletion of CCL5 and/or CCL20 from Th17-derived supernatants was obtained by specific capture antibodies (R&D Systems). Cell migration was quantified by flow cytometry.

Amicarella F., Muraro M.G., Hirt C., Cremonesi E., Padovan E., Mele V., Governa V., Han J., Huber X., Droeser R.A., Zuber M., Adamina M., Bolli M., Rosso R., Lugli A., Zlobec I., Terracciano L., Tornillo L., Zajac P., Eppenberger-Castori S., Trapani F., Oertli D, & Iezzi G. (2015). Dual role of tumour-infiltrating T helper 17 cells in human colorectal cancer. Gut, 66(4), 692-704.

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Isolation and Culture of Bone Marrow Cells

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To harvest bone marrow cells, femurs and tibiae from WT BL6 mice were flushed under sterile conditions. Cells were filtered through a 40 μm mesh and cultured in Teflon bags (PermaLife PL-30) for 6 d in DMEM supplemented with 5% FBS, or 5% serum derived from either LF/HF mice or lean/obese humans. Recombinant proteins (CCL25, CD40L, GM-CSF, IGFBP2, IL5, IL6, MMP3, MMP9, 100 ng ml⁻¹ for all; R&D Systems) or neutralization antibodies (IgG versus anti-GM-CSF, 0.1 μg ml⁻¹) were added as indicated. Treatment media were changed every other day. After 6 d, flow cytometry was performed using antibodies against murine CD45, CD11b and Gr1 (see Supplementary Table 3).

Quail D.F., Olson O.C., Bhardwaj P., Walsh L.A., Akkari L., Quick M.L., Chen I.C., Wendel N., Ben-Chetrit N., Walker J., Holt P.R., Dannenberg A.J, & Joyce J.A. (2017). Obesity alters the lung myeloid cell landscape to enhance breast cancer metastasis through IL5 and GM-CSF. Nature cell biology, 19(8), 974-987.

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