Four AML cell lines, MOLM-13 (AML M5), KASUMI-1 (AML M2), OCI-AML3 (AML M4) and NOMO-1 (AML M5) were obtained from the American Type Culture Collection, and were mycoplasma-tested and authenticated using the LGC Standards Cell Line Authentication service. The cell lines were cultured at 37°C in a 5% CO2 atmosphere at a density of 0.3×106 cells/ml in complete medium, in T75 flasks. MOLM-13 and KASUMI-1 cells were cultured in RPMI-1640 (Euroclone) supplemented with 20% heat-inactivated FBS (GE Healthcare), 2 mM L-glutamine (GE Healthcare), 100 U/ml penicillin, 100 µg/ml streptomycin (GE Healthcare) and 0.2% Mycozap (Lonza, Inc.). OCI-AML3 cells were cultured in α-MEM (Lonza, Inc.) with 20% FBS, 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin. NOMO-1 cells were grown in RPMI-1640 with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin.
Heat inactivated fbs
Manufactured by GE Healthcare
Sourced in United States
Heat-inactivated FBS is a laboratory reagent used as a supplement in cell culture media. It is derived from fetal bovine serum and has undergone a heat-inactivation process to deactivate any potential biological contaminants.
Automatically generated - may contain errors
Lab products found in correlation
L glutamine, by Lonza (2 mentions)
Penicillin, by GE Healthcare (1 mentions)
Streptomycin, by GE Healthcare (1 mentions)
L glutamine, by GE Healthcare (1 mentions)
Rpmi 1640, by Euroclone (1 mentions)
α mem, by Lonza (1 mentions)
Penicillin, by Lonza (1 mentions)
Streptomycin, by Lonza (1 mentions)
Mycozap, by Lonza (1 mentions)
Phorbol 12, by Merck Group (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Merck Group (1 mentions)
β mercaptoethanol, by Merck Group (1 mentions)
Phytohemagglutinin (pha), by Merck Group (1 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions)
Non essential amino acids (neaa), by Lonza (1 mentions)
Histopaque 1077, by Merck Group (1 mentions)
Hepes, by Lonza (1 mentions)
5 protocols using heat inactivated fbs
1
Culturing Four AML Cell Lines
2
Differentiation of THP-1 Cells into Macrophages
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THP-1 cells provided by ATCC were cultured as previously described (43 (link)). Cells were subcultured at a density of 5 × 104 cells/cm2 on 24-well clusters with 12-mm round cover glasses. Cells were differentiated to macrophages with phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich) at a concentration of 100 nmol/L in RPMI 1640 (Sigma-Aldrich) supplemented with 10% heat-inactivated FBS (GE Healthcare Life Sciences), 50 µM β-mercaptoethanol (Sigma-Aldrich), and penicillin (100 U/mL)/streptomycin (100 µg/mL) (Life Technologies) for 3 d. Differentiation of PMA-treated cells was enhanced after the initial 3-d stimulus by removing the PMA-containing media and then incubating the cells in fresh supplemented RPMI 1640 for a further 3 d.
3
Generating Bone Marrow-Derived Dendritic Cells
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Bone marrow-derived DCs from C57BL/6 mice were generated as described previously [28 (link)] and finally resuspended at a density of 2 × 106 cells in 10-mL R10 medium consisting of RPMI1640, 1% penicillin/streptomycin/L-glutamine, 2-ME and 10% heat-inactivated FBS (GE Healthcare, Chicago, Illinois, United States), additionally supplemented with GM-CSF supernatant (1:10) from a cell line stably transfected with the murine GM-CSF [29 (link)]. At days 3 and 6, 10 mL of fresh R10 supplemented with GM–CSF supernatant (1:10) was added, with removing 50% of the old cell culture supernatant at day 6 before. Maturation of BMDCs was induced at day 7 by the addition of 0.1 ng/mL LPS for 20 h. At day 8, cells were used for further experiments.
4
Lymph Node and PBMC Activation for HIV Infection
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Lymph node cells were obtained by mechanical separation of lymph nodes and frozen at 5 × 106 cells/ml in a solution of 90% FBS and 10% DMSO with 2.5 μg/ml Amphotericin B (Lonza). Cells were stored in liquid nitrogen until use, then thawed and resuspended at 106 cells/ml in complete RPMI 1640 medium supplemented with L-Glutamine, sodium pyruvate, HEPES, non-essential amino acids (Lonza), 10% heat-inactivated FBS (Hyclone), and IL-2 at 5 ng/ml (PeproTech). Phytohemagglutinin at 10 µg/ml (Sigma-Aldrich, St Louis, MO) was added to activate cells. PBMCs were isolated by density gradient centrifugation using Histopaque 1077 (Sigma-Aldrich) and cultured at 106 cells/ml in complete RPMI 1640 medium supplemented with L-Glutamine, sodium pyruvate, HEPES, non-essential amino acids (Lonza), 10% heat-inactivated FBS (GE Healthcare Bio-Sciences, Pittsburgh, PA), and IL-2 at 5 ng/ml (PeproTech, Rocky Hill, NJ). Phytohemagglutinin at 10 µg/ml (Sigma-Aldrich) was added to activate cells. For both primary cell types, donor cells for coculture infection were cultured for one day then infected by cell-free virus, while target cells were cultured for three days and infected with either cell-free HIV or infected donor cells.
5
Culturing Adherent and Suspension Cell Lines
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RPMI 1640 culture medium (with L-glutamine, Gibco) was supplemented with 10% v/v heat-inactivated FBS (standard quality, EU approved, GE Healthcare), 1% v/v penicillin- streptomycin (Sigma-Aldrich), 1% v/v HEPES Buffer (1 M, Lonza), 1% v/v sodium pyruvate solution (100 mM, Sigma-Aldrich) and 500 nM β2-Mercaptoethanol (Sigma-Aldrich). Adherent K89 and suspension B3Z were cultured in complete RPMI at 37 °C, 5% CO 2 and harvested as necessary to keep them in their log phase. 1 mM EDTA (Biowhittaker) was added in the passaging of K89, and the population was gently detached from the flasks mechanically, using a Pasteur pipette. Limiting dilution sub-cloning was initially used on the lymphocytes population to select for cells that were highly sensitive to SIINFEKL (SL8) epitope, and sensitivity assays were periodically run to ensure the functionality of the cell model (see ESI † 2).
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