Human gm csf

Manufactured by Miltenyi Biotec

Sourced in United Kingdom

Human GM-CSF is a recombinant cytokine that stimulates the production and function of granulocytes and macrophages. It is a key regulator of hematopoiesis and immune function.

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GM-CSF (180 citations) Recombinant human GM-CSF (22 citations) Recombinant human granulocyte macrophage colony-stimulating factor (GM-CSF) (7 citations) Human granulocyte macrophage colony‐stimulating factor (GM‐CSF), (2 citations)

11 protocols using human gm csf

Isolation and Culture of Primary Human Cells

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293T and MAGI cells were maintained as previously described [45 (link)]. Primary human monocytes and CD4⁺ T cells were isolated from peripheral blood buffy coats by positive selection using CD14 or CD4 beads (Miltenyi Biotec, San Diego, CA), as previously described [46 (link)], and maintained in RPMI medium with 10 % FCS and penicillin/streptomycin. CD14⁺ monocytes were matured to macrophages using 5 ng/mL human GM-CSF for 7 days (Miltenyi Biotec). CD4⁺ T cells were activated with 5 µg/mL phytohemagglutinin (Sigma-Aldrich) and 20 units/mL IL2 (Miltenyi Biotec) for 1 day, then 20 units/mL IL2 alone for 5 days.

Daly M.B., Roth M.E., Bonnac L., Maldonado J.O., Xie J., Clouser C.L., Patterson S.E., Kim B, & Mansky L.M. (2016). Dual anti-HIV mechanism of clofarabine. Retrovirology, 13, 20.

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Macrophage Activation by IgA-opsonized S. aureus

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Peripheral blood mononuclear cells were isolated from blood samples by density gradient centrifugation over Ficoll-Paque (Eurobio). Monocytes for macrophage differentiation were isolated by plastic adherence as follows: peripheral blood mononuclear cells were distributed into a 25-cm² flask and allowed to adhere for 1 h at 37°C in RPMI (Gibco) supplemented with 10% FCS (Biowest). Nonadherent cells were removed, and the adherent monocytes were washed twice with medium, detached with Versene (Invitrogen), counted, seeded in 96-well U-bottom plates at a density of 5.10⁴ cells/well, and cultured in RPMI-10% FCS in the presence of 100 ng/ml human GM-CSF (Miltenyi Biotec) in a total volume of 100 µl. At day 6, macrophages were stimulated with 5.10⁵S. aureus, which had been preincubated with 0.5 mg/ml of purified IgA1 or IgA2 and washed. After 24 h, cytokine levels in supernatants were measured using the Cytokine 3-Plex A (IL-6, IL-10, and TNF-α) immunoassay (Quanterix) relying on a Single Molecule Array (Simoa) technology run on a HD-1 Analyzer (Quanterix). Working dilutions were 1:500 in diluent sample. The lower limits of quantification for IL-6, IL-10, and TNF-α were 5.5, 3.65, and 25.5 pg/ml, respectively.

Sterlin D., Fadlallah J., Adams O., Fieschi C., Parizot C., Dorgham K., Rajkumar A., Autaa G., El-Kafsi H., Charuel J.L., Juste C., Jönsson F., Candela T., Wardemann H., Aubry A., Capito C., Brisson H., Tresallet C., Cummings R.D., Larsen M., Yssel H., von Gunten S, & Gorochov G. (2019). Human IgA binds a diverse array of commensal bacteria. The Journal of Experimental Medicine, 217(3), e20181635.

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Isolation and Differentiation of Primary Human Monocytes

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Primary human monocytes were isolated from the peripheral blood buffy coats by positive selection using MACS CD14⁺ beads as previously described (Hollenbaugh et al., 2014 (link)). Monocytes were maturated into monocyte-derived macrophages (MDMs) in the presence of 5 ng/ml human GM-CSF (Miltenyl Biotec) treated at days 0 and 2 of maturation. MDMs were used at day 7 or afterwards for experiments. For the HIV-1 RT activity assay, 250,000 monocytes were maturated in 24-well plates. For the metabolomics analysis, 2 million monocytes were maturated in 3 cm dishes (Hollenbaugh et al., 2011 (link)). To determine dNTP levels using the HIV-1 RT-based assay (Diamond et al., 2004 (link)), 1 million monocytes were maturated in 6-well plates.

Hollenbaugh J.A., Montero C., Schinazi R.F., Munger J, & Kim B. (2016). Metabolic profiling during HIV-1 and HIV-2 infection of primary human monocyte-derived macrophages. Virology, 491, 106-114.

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Isolation and Culture of Human Dendritic Cells

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Human peripheral blood mononuclear cells (PBMCs) were collected from healthy donors’ buffy coat (provided by the University Hospitals of Geneva (HUG)) using Ficoll-Paque Plus (Cytiva). Monocytes were isolated using the Classical Monocyte Isolation Kit (human, Miltenyi Biotec) according to the manufacturer’s instructions. Cells were centrifuged and resuspended in complete medium (RPMI, 10% heat-inactivated FCS, 2 mM l-glutamine, 1% penicillin–streptomycin, 50 µM β-mercaptoethanol) plus 500 U ml⁻¹ human GM-CSF and 250 U ml⁻¹ human IL-4 (both from Miltenyi Biotec) and cultured in 12-well cell culture plates at 37 °C with 5% CO₂. The medium was refreshed every other day. On day 6, non-adherent cells were collected for experiments.

Wang C., Barnoud C., Cenerenti M., Sun M., Caffa I., Kizil B., Bill R., Liu Y., Pick R., Garnier L., Gkountidi O.A., Ince L.M., Holtkamp S., Fournier N., Michielin O., Speiser D.E., Hugues S., Nencioni A., Pittet M.J., Jandus C, & Scheiermann C. (2022). Dendritic cells direct circadian anti-tumour immune responses. Nature, 614(7946), 136-143.

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Monocyte Isolation and Macrophage Differentiation

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Monocytes were isolated from whole blood (New York Blood Service, Long Island New York) by using MACS^® CD14⁺ beads as described previously [33 (link)] and cultured in the presence of 5 ng/mL human GM-CSF (Miltenyi Biotec). MDMs were utilized at day 7 of maturation for experiments.

Hollenbaugh J.A., Shelton J., Tao S., Amiralaei S., Liu P., Lu X., Goetze R.W., Zhou L., Nettles J.H., Schinazi R.F, & Kim B. (2017). Substrates and Inhibitors of SAMHD1. PLoS ONE, 12(1), e0169052.

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Generation of Monocyte-Derived Microglia and Macrophages

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To generate monocyte-derived microglia (MDMG), CD14⁺ peripheral blood monocytes positively isolated with CD14 microbeads (Miltenyi Biotec) (68 (link)) were seeded on Geltrex-coated (Gibco) tissue culture plates and cultured for 12 to 14 days in RPMI 1640 Glutamax (Gibco) supplemented with 1% pen/strep, 100 µg/ml of IL-34 (PeproTech), and human GM-CSF (10 ng per ml; Miltenyi Biotec). Human monocyte-derived macrophages (MDMs) were derived from CD14⁺ peripheral blood monocytes by culturing in RPMI 1640 (Gibco) containing 10% heat-inactivated human AB serum (Sigma-Aldrich), and recombinant human M-CSF (20 ng per ml; PeproTech) for 5 to 6 days and maintained in the same medium.

Akiyama H., Jalloh S., Park S., Lei M., Mostoslavsky G, & Gummuluru S. (2021). Expression of HIV-1 Intron-Containing RNA in Microglia Induces Inflammatory Responses. Journal of Virology, 95(5), e01386-20.

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Isolation and Culture of Primary Monocytes and CD4+ T Cells

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Monocytes were isolated from whole blood (New York Blood Service, Long Island New York) by using MACS^® CD14⁺ beads as described previously (Hollenbaugh et al., 2013 (link)) and cultured in the presence of 5 ng/ml human GM-CSF (Miltenyi Biotec). The resulting MDMs were utilized on day 7 for all experiments. From monocyte-depleted PBMCs, MACS CD4 beads were used to enrich for CD4⁺ T cells as previously described (Hollenbaugh et al., 2011 (link)). Cells were cryopreserved (10 million cells/ml, −80 °C) until reanimated for experimentation. Upon reanimation in DMEM (5% FBS/30 million cells), 60–120 million CD4⁺ T cells were activated using 5 μg/ml of PHA (Sigma) and 10 ng/ml IL-2 (NIH AIDS Reagent Program). On day 3 post-reanimation, fresh medium and 10 ng/ml IL-2 were added to cells and cells were permitted to expand for a further 48 h after which cells were utilized in experiments.

Hollenbaugh J.A., Schader S.M., Schinazi R.F, & Kim B. (2015). Differential regulatory activities of viral protein X for anti-viral efficacy of nucleos(t)ide reverse transcriptase inhibitors in monocyte-derived macrophages and activated CD4+ T cells. Virology, 485, 313-321.

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Culturing Vero, C6/36, and Human Monocyte-Derived Dendritic Cells

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Vero cells (CCL-81) were obtained from ATCC and maintained in DMEM (Gibco) supplemented with 10% fetal bovine serum (Biochrom). A. albopictus C6/36 cells were obained from ATCC and cultured in G-MEM-BHK-21 (Gibco) supplemented with 2% FBS (Biochrom). Human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats of anonymous healthy blood donors (Interregional blood transfusion SRC Ltd, Bern) by density centrifugation over Ficoll-Paque™ PLUS (GE healthcare). Selection of CD14+ monocytes was done using coated magnetic beads according to the manufacturer’s instructions (Miltenyi Biotec) and seeded at a rate of 1 × 10⁶ cells/ml in RPMI 1640 (Gibco) supplemented with 10% FBS (Gibco), GlutaMax (Gibco), penicillin-streptomycin (Gibco), human GM-CSF (100 ng/ml) and human IL-4 (40 ng/ml) (both from Miltenyi Biotec) for six days at 37 °C, 5% CO₂. The cultures were fed with fresh cytokines at day three post-isolation.

Vielle N.J., Zumkehr B., García-Nicolás O., Blank F., Stojanov M., Musso D., Baud D., Summerfield A, & Alves M.P. (2018). Silent infection of human dendritic cells by African and Asian strains of Zika virus. Scientific Reports, 8, 5440.

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Isolation and Differentiation of Human Myeloid Cells

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Human monocyte-derived macrophages (MDMs) and monocyte-derived dendritic cells (MDDCs) were derived from beads-isolated CD14⁺ peripheral blood monocytes^{47 (link)} by culturing in RPMI1640 (Invitrogen) containing 10% heat-inactivated human AB serum (Gemini Bio Products, Sigma or Corning) and recombinant human M-CSF (20 ng ml^–1; Peprotech) or containing 10% heat-inactivated fetal bovine serum supplemented with human GM-CSF (10 ng ml^–1, Miltenyi) and IL-4 (1000 U ml^–1, BD), respectively, for 5–6 days. HEK293T (ATCC), 293 ISRE-luc (Drs. Junzhi Wang and Xuguang Li), HeLa (NIH AIDS Reagent Program) and TZM-bl (NIH AIDS Reagent Program) were maintained in DMEM (Invitrogen) containing 10% heat-inactivated FBS (Invitrogen)^{49 ,53 (link),54 (link)}. All cell lines have been tested for mycoplasma contamination and confirmed negative. Cell viability was measured by a conventional MTT assay.

Akiyama H., Miller C.M., Ettinger C.R., Belkina A.C., Snyder-Cappione J.E, & Gummuluru S. (2018). HIV-1 intron-containing RNA expression induces innate immune activation and T cell dysfunction. Nature Communications, 9, 3450.

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Differentiation and Activation of Human Myeloid Cells

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Human myeloid cell line U937 was obtained from the biological resource center Cellulonet (Lyon, France) and grown in RPMI 1640 GlutaMax (Thermo Fisher Scientific) supplemented with 10% (vol/vol) heat-inactivated fetal calf serum (Dutscher), 10 mM Hepes, pH 7.5 (Thermo Fisher Scientific), and 0.04 mg/ml gentamycin (Thermo Fisher Scientific). U937 cells were exposed to 100 ng × ml⁻¹ of PMA (InvivoGen) for differentiation. For primary macrophage differentiation, purified monocytes were plated in the presence of 50 ng/ml M-CSF (Miltenyi Biotec) and incubated at 37°C in 5% CO₂ for times indicated in the figure legends. Human GM-CSF and human IL-4 were from Miltenyi Biotec. Rapamycin was from Tocris. Torin 2 and BafA1 were from Invivogen. The inhibitors SB203580 (p38 inhibitor), SP600125 (JNK inhibitor), PD98059 (ERK inhibitor), and LY294002 (PI3K inhibitor) from InvivoGen and AZD5363 (AKT inhibitor) from Selleckchem were gifts from Patrice André and the Vincent Lotteau Laboratory (Centre International de Recherche en Infectiologie, Lyon, France). Chloroquine, pepstadin A, and E64D were from Sigma-Aldrich, while BafA1 was from Invivogen. LPS used for macrophages stimulation was from Invivogen. Heat-killed S. enterica serovar Typhimurium (strain SL1344) and heat-killed S. aureus (strain Lug960) were cultured in T. Henry’s laboratory.

Omarjee O., Mathieu A.L., Quiniou G., Moreews M., Ainouze M., Frachette C., Melki I., Dumaine C., Gerfaud-Valentin M., Duquesne A., Kallinich T., Tahir Turanli E., Malcus C., Viel S., Pescarmona R., Georgin-Lavialle S., Jamilloux Y., Larbre J.P., Sarrabay G., Magnotti F., Rice G.I., Bleicher F., Reboulet J., Merabet S., Henry T., Crow Y.J., Faure M., Walzer T, & Belot A. (2021). LACC1 deficiency links juvenile arthritis with autophagy and metabolism in macrophages. The Journal of Experimental Medicine, 218(3), e20201006.

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