To evaluate the biological activity of compounds 11 –14 , three human tumor cell lines (European Collection of Cell Culture, Salisbury, Wiltshire, UK) were used: A375-C5 (melanoma), MCF-7 (breast adenocarcinoma) and NCI-H460 (non-small cell lung cancer). Cells were grown in RPMI-1640 culture medium (Biochrom, Berlin, Germany) supplemented with 5% heat-inactivated fetal bovine serum (FBS, Biochrom) and maintained in a humidified incubator at 37 °C with 5% CO2 (Hera Cell, Heraeus).
Rpmi 1640 culture medium
Manufactured by Harvard Bioscience
Sourced in Germany
RPMI 1640 is a cell culture medium designed for the in vitro cultivation of a variety of cell types, including human and animal cells. It provides essential nutrients, amino acids, vitamins, and other components required for cell growth and maintenance.
Automatically generated - may contain errors
Lab products found in correlation
Fetal calf serum (fcs), by Harvard Bioscience (3 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Interleukin 4 (il 4), by R&D Systems (1 mentions)
Q vd oph hydrate, by Merck Group (1 mentions)
Soluble anti cd28, by BD (1 mentions)
Anti cd3, by Thermo Fisher Scientific (1 mentions)
Cell culture flasks and plates, by Greiner (1 mentions)
Biocoll separating solution, by Harvard Bioscience (1 mentions)
Penicillin streptomycin, by Harvard Bioscience (1 mentions)
5 protocols using rpmi 1640 culture medium
1
Evaluation of Anticancer Compounds
2
CFSE Labeling and B Cell Stimulation
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Isolated B cells were labeled with 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester (CFSE, Invitrogen, Carlsbad, CA, USA) for 10 min at 37°C. Labeled B cells were washed two times with RPMI 1640 culture medium (Biochrom, Berlin, Germany) with supplements (10% FCS, Biochrom, Berlin, Germany). B cells were cultured in 96-well flat-bottom tissue culture plates with either 100 ng/mL LPS or 10 ng/mL Il-4 (R&D Systems, Minneapolis, MN, USA) and 2 μg/mL CD40-L (2 × 105 cells/well). Cells were cultured at 37°C in humidified 5% CO2 incubator for 72 h prior to analysis by flow cytometry.
3
T Cell Proliferation Assay
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Purified T cells were cultured in RPMI 1640 culture medium (Biochrom, Berlin, Germany) with supplements (10% FCS, Biochrom) in 96-well flat-bottom tissue culture plates precoated with 5 μg/ml anti-CD3 (eBioscience, San Diego, CA, USA), 2 μg/ml soluble anti-CD28 (BD Pharmingen, Franklin Lakes, NJ, USA) (2 × 105 cells/well), and 10 μM Q-VD-OPh hydrate (Sigma, St. Louis, MO, USA) where indicated. Cells were cultured at 37 °C in a humidified 5% CO2 incubator for 72 h. For proliferation analysis, isolated T cells were labeled with 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester (CFSE, Invitrogen, Carlsbad, CA, USA) for 10 min at 37 °C and washed twice with culture medium.
4
AhR Ligand Assay in Salmonella
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All chemicals or reagents were obtained from Sigma Aldrich (Taufkirchen, Germany) unless otherwise noted. Cell culture flasks and plates were purchased from Greiner Bio-One (Frickenhausen, Germany). RPMI 1640 culture medium (Biochrom, Berlin, Germany) was supplemented with 10 mM HEPES buffer, 2 mM L-glutamine, 10% (v/v) fetal calf serum (FCS) and 100 U/mL penicillin, 100 µg/mL streptomycin and 50 μM β-mercaptoethanol to be used as complete cell culture medium. Salmonella enterica growth experiments were carried out using Luria Bertani (LB) fluid medium or agar (both from Becton Dickinson, Heidelberg, Germany) or selective Xylose-Lysine-Desoxicholate (XLD) agar (Heipha Dr. Müller, Eppelheim, Germany). For in vivo experiments, the AhR ligands BaP and FICZ (Enzo Life Science, Lörrach, Germany) were dissolved in corn oil. For Griess reagent, 0.1% N-(1 naphthyl)-ethylenediamine-dihydrochloride was solved in ethanol (>99.8%) and mixed with a solution of 1% sulfanilamide solved in 5% phosphoric acid.
5
Isolation and Functional Analysis of NK Cells
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Fresh heparinized peripheral blood samples were collected from 100 individuals, 33 to 87 years of age, including males and females between 2012 and 2015. PBMCs were isolated from 20 mL of heparinized blood by density gradient centrifugation using Biocoll separating solution (Biochrom GmbH, Berlin, Germany). PBMCs were washed with phosphate-buffered saline (PBS) and resuspended in complete RPMI 1640 culture medium (Biochrom GmbH), supplemented with 1% penicillin/streptomycin (Biochrom GmbH; final concentrations 100 U/mL and 100 µg/mL, respectively) and 10% fetal calf serum (Biochrom GmbH). PBMCs were adjusted to 1x106 cells/mL. Measurement of NK cell function was performed in cell culture laboratories of GANZIMMUN Diagnostics AG (Mainz, Germany).
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