The human AML cell lines (THP1, NOMO1, Kasumi-1 and SKNO-1) were obtained from State Key Laboratory of Experimental Hematology. THP1, NOMO1 and Kasumi-1 cells were cultured at 1-2 × 106 cells/mL in RPMI-1640 (C11875500BT, GIBCO, USA) containing 10% fetal bovine serum (FBS, C04001, Vivacell, China), 1% penicillin/streptomycin (SV30010, HyClone, USA). In addition to the above components, cell culture medium for SKNO-1 was supplemented with 10 ng/ml GM-CSF (300-03, PeproTech, USA). Cells were maintained at 37°C in a humidified incubator (ThermoFisher, USA).
Gm csf 300 03
Manufactured by Thermo Fisher Scientific
Sourced in United States
GM-CSF (300-03) is a recombinant human Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) protein produced in E. coli. GM-CSF is a cytokine that stimulates the production and function of granulocytes and macrophages.
Automatically generated - may contain errors
Lab products found in correlation
Humidified incubator, by Thermo Fisher Scientific (1 mentions)
Kasumi 1, by Thermo Fisher Scientific (1 mentions)
Skno 1, by Thermo Fisher Scientific (1 mentions)
Nomo 1, by Thermo Fisher Scientific (1 mentions)
Thp 1, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Tnf α 210 ta cf, by R&D Systems (1 mentions)
P1585 1mg, by Merck Group (1 mentions)
Histopaque, by Merck Group (1 mentions)
4 protocols using gm csf 300 03
1
Culture of Human AML Cell Lines
2
Generation of Dendritic Cells from Human Serum
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Very low endotoxin RPMI 1640 medium (FG 1415; Biochrom) supplemented with 1.5% human serum (serum pool of AB positive adult males; Institute for Transfusion Medicine)—hereafter named DC medium—was used for the generation of DCs and all coculture experiments. The following reagents were used to generate DCs: GM-CSF (300-03), rhIL-4 (200-04), IFN-γ (300-02; all PeproTech), rhIL-1β (201-LB), TNF-α (210-TA/CF; both R&D Systems), PGE2 (P5640; Sigma-Aldrich), and R848 (tlrl-r848; InvivoGen).
3
Macrophage Differentiation and CAR Expression
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THP-1 cells were stimulated to differentiate into human macrophages using 100 ng/ml PMA (P1585-1MG, Sigma), and the stimuli were evaluated using CD11b and CD68 expression for subsequent experiments. CAR adenovirus was purchased from the OBiO Technology Company Limited (Shanghai, China). CAR THP-1 cells targeting CD47 and HER2 and vehicle control cells were prepared by adenovirus infection of PMA-stimulated THP-1 cells according to the manufacturer’s instructions. After infection, CAR THP-1 cells and vehicle control cells were cultured in RPMI1640 supplemented with 10% FBS and 100 ng/ml recombinant human granulocyte monocyte colony-stimulating factor (GM-CSF, 300-03, PeproTech). After three days, the cells were analyzed by flow cytometry for Green fluorescent protein (GFP) expression to determine CAR expression.
4
Isolation and Culture of Human Monocytes
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The following collection of human blood has been approved by the Tasmanian Health and Medical Human Research Ethics Committee (H0016505). Peripheral blood mononuclear cells (PMBCs) were isolated from six healthy adults aged between 20 and 50 years of age. Briefly, 32 mL of blood was collected from each patient using EDTA-BD Vacutainers (366643; Becton Dickinson, Franklin Lakes, NJ, USA). Samples were incubated at room temperature for 20 min with gentle mixing. Blood was aseptically layered onto 4 mL of Histopaque (10771; Sigma-Aldrich, St. Louis, MO, USA) in a 1:1 ratio before being centrifuged at 100 g for 30 min. Density gradient separation allowed the isolation of mononuclear cells which were then washed twice by centrifugation at 300 g. Cells were resuspended in serum-free RPMI-1640 and seeded at a density of 6 × 106 cells/mL. Cells were incubated for 1 h at 37 °C in a humidified atmosphere of 5% CO2 and then washed twice to remove non-adherent cells, providing pure monocyte cultures. RPMI-1640 media containing 10% FBS was supplemented with 2 ng/mL of granulocyte-macrophage colony-stimulating factor (GM-CSF 300-03; Pepro Tech Inc., Rocky Hill, NJ, USA) for 12 days, refreshing every 3 days.
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