Monocytes were purified from blood by Ficoll (Eurobio, Coutaboeuf, France) density gradient separation and by negative selection using the EasySep™ Human Monocyte Enrichment Kit (Stem Cell, Grenoble, France). Monocytes were suspended in RPMI 1640 medium (Dutscher, Brumate, France) supplemented with 10% heat-inactivated fetal calf serum (FCS; Dutscher, Brumate, France), 1% l -glutamine (Life Technologies, Carlsbad, CA, USA), and 0.2 μg/mL of recombinant human macrophage colony stimulating factor (rh-M-CSF, Immunotools, Friesoythe, Germany). Cells were seeded into 48-well culture plates at a density of 2.5 × 105 and were incubated at 37 °C in a humidified 5% CO2 atmosphere for six days.
Rpmi 1640 medium
Manufactured by Dutscher
Sourced in France
RPMI 1640 medium is a commonly used cell culture medium formulation. It is a balanced salt solution that provides essential nutrients and growth factors to support the in vitro cultivation of a variety of cell types, including mammalian, insect, and plant cells.
Automatically generated - may contain errors
Lab products found in correlation
L glutamine, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions)
Ficoll, by Eurobio Scientific (1 mentions)
Easysep human monocyte enrichment kit, by STEMCELL (1 mentions)
Rh m csf, by Immunotools (1 mentions)
Fetal calf serum (fcs), by Dutscher (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Foetal bovine serum (fbs), by Dutscher (1 mentions)
B16f10 mouse melanoma cells, by American Type Culture Collection (1 mentions)
A549 human lung cancer cells, by American Type Culture Collection (1 mentions)
Mrc 5 human lung fibroblasts, by American Type Culture Collection (1 mentions)
Sk mel 2 human melanoma cells, by American Type Culture Collection (1 mentions)
Wm266 4, by Thermo Fisher Scientific (1 mentions)
α mem medium, by Thermo Fisher Scientific (1 mentions)
Lsr 2 cytometer, by BD (1 mentions)
Transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
Ficoll hypaque, by GE Healthcare (1 mentions)
Foetal calf serum, by PAN Biotech (1 mentions)
8 protocols using rpmi 1640 medium
1
Isolation and Differentiation of Human Monocytes
2
Cell Culture Protocols for HL60 and H1975 Cell Lines
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HL60 cells were cultured in RPMI-1640 medium (LifeTechnologies) supplemented with 10% of heat-inactivated Fetal Calf Serum (FCS) (lot n°S11060S181, Dominique Dutscher) containing Penicillin-Streptomycin (50 UI/ml and 50 µg/ml) (GIBCO®). HL60 cells were seeded every 2–3 days at 100,000 cells/mL in 5 mL in 25 cm2 flasks (BD Falcon). H1975 cells were cultured in RPMI-1640 medium supplemented with 10% Fetal Calf Serum (FCS) (lot n°S11060S181, Dominique Dutscher) and 1% Sodium Pyruvate and Hepes (Lifetechnologies). H1975 cells were seeded twice a week at 300,000 cells/mL in 20 mL in a 75 cm2 flask (BD Falcon). All cell lines were incubated at 37 °C and 5% CO2.
3
Cell Culture of Lung and Melanoma Cells
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Non-cancerous human lung cells (MRC-5) human lung fibroblasts, A549 human lung cancer cells, B16F10 mouse melanoma cells and SK-MEL-2 human melanoma cells were purchased from the ATCC. All cell lines were cultured at 37°C, 5% CO2 in RPMI 1640 medium (Dutscher) supplemented with 10% foetal bovine serum (Dutscher) depleted in exosomes and were tested weekly for mycoplasma contamination.
4
Optimizing S100-EPISPOT Assay for Melanoma
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The melanoma cancer cell lines WM-266-4 (ATCC® CRL-1676™) and MV3 (kindly provided by Klaus Pantel, University of Tumor Biology, Hamburg, Germany) were used for optimizing the S100-EPISPOT assay. WM-266-4 cells were maintained in αMEM medium (22571, Gibco, Grand Island, USA) supplemented with 10% fetal calf serum (FCS), and MV3 cells in RPMI 1640 medium (L0501, Dominique Dutscher, Brumath, France), supplemented with 5mM L-glutamine (25030, Gibco, Grand Island, USA) and 10% FCS.
5
Isolation and Characterization of PBMCs
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Healthy donors PBMC were obtained after Ficoll-Hypaque (GE Healthcare) density centrifugation and cultured in RPMI 1640 medium (Dutscher) supplemented with 10% fetal bovine serum (ThermoFisher Scientific). Human tumor biopsy specimens were weighed, washed with PBS and mechanically disrupted. These preparations were centrifuged on a Ficoll gradient and frozen. When specified in text, T cells were isolated by negative selection using the Human T Lymphocytes Enrichment Set-DM (BD Biosciences) according to the manufacturer’s instructions. TC1 cells expressing the HPV 16 E6 and E7 proteins were developed in the laboratory of T.C. Wu (Johns Hopkins University). Resting PBMC control was processed for immunostaining with the specified antibodies or ADT (Biolegend) for 20 min at room temperature. For intracellular stainings, cells were fixed and permeabilized using the transcription factor staining buffer set (eBioscience) before staining with the proper antibodies or reagent. Used antibodies are listed on the Key Resource Table. Flow cytometry acquisitions were performed on a BD LSR II cytometer (BD Biosciences) and data were analyzed with Cytobank (http://www.cytobank.org ).
6
Preparation of AR42J Cell Membranes
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The SST2-expressing AR42J, acinar pancreatic tumour cell line derived from a transplantable tumour of a rat exocrine pancreas, was provided by ATCC (LGC Standards, Molsheim, France). Cells were cultured in RPMI 1640 medium (Dutscher, Bernolsheim, France), supplemented with 10% foetal calf serum (PAN-Biotech, Aidenbach, Germany), in a humidified atmosphere at 37 °C with 5% CO2. AR42J membranes were obtained by sonication in 50 mM Tris–HCl, pH 7.4, and centrifugation at 39,000× g for 10 min at 4 °C. The pellet was resuspended in the same buffer and centrifuged at 50,000 ×g for 10 min at 4 °C, and membranes in the resulting pellet were stored at −80 °C until further use.
7
Melanoma Culture Protocol
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A panel of different patient-derived melanoma cultures was obtained from the laboratory of Prof. M. Levesque from Dermatology, USZ (cf Table 1 ). Two more cell lines from the laboratory of L. Sommer, Anatomy, UZH were obtained; these cell lines harbor a mutation in β-catenin (pS37F). Cells were cultured in RPMI-1640 medium (L0998-500, Dominique Dutscher) enriched with 10% FCS (S181B-500, Dominique Dutscher), L-Glutamine, and Na-Pyruvat (11360-039, Gibco) (1% each).
8
Transfection of A549 and SiHa Cell Lines
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The A549 (NSCLC) cell line was obtained from Dr. Christophe Borg (INSERM UMR1098, Besançon, France) and the SiHa cells were obtained from ATCC (HTB-35™). A549 cells were grown in Dulbecco’s minimum essential medium (DMEM) 1 g/L glucose (L0066, Dominique Dutscher, Brumath, France) and SiHa cells were cultured in RPMI1640 medium (Dutscher, L0498). Both media were supplemented with fetal bovine serum (10%) (S1810, Dominique Dutscher). Cells were cultured at 37 °C in 5% CO2 and routinely used at 70–80% confluence.
Transfections of the plasmids were performed using the Lipofectamine 2000 reagent (11668030, ThermoFisher) following the supplier’s recommendations. Briefly, cells were seeded in 6-well plates and transfected for 24 h with 1 µg of plasmid and 2 µL of Lipofectamine 2000 reagent and harvested 24 h after the end of the transfection. Transfections of the siRNA were conducted using the same protocol with 20 pmol of siRNA and 3 µL of Lipofectamine 200 reagent.
Transfections of the plasmids were performed using the Lipofectamine 2000 reagent (11668030, ThermoFisher) following the supplier’s recommendations. Briefly, cells were seeded in 6-well plates and transfected for 24 h with 1 µg of plasmid and 2 µL of Lipofectamine 2000 reagent and harvested 24 h after the end of the transfection. Transfections of the siRNA were conducted using the same protocol with 20 pmol of siRNA and 3 µL of Lipofectamine 200 reagent.
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