Five human tumor cell lines, obtained from the European Collection of Cell Culture, were used in this study, namely A375-C5 derived from melanoma, MCF-7 derived from breast adenocarcinoma, NCI-H460 derived from non-small cell lung adenocarcinoma, and HCT-15 and Caco-2 derived from colorectal carcinoma. The HPAEpic cell line (Human Pulmonary Alveolar Epithelial Cells) was also included in the screening assays and was obtained from ScienCell Research Laboratories. All cell lines were maintained at 37 °C in a 5% CO2 humidified incubator, and all experiments were performed when exponentially growing cells presented more than 95% viability. A375-C5, MCF-7, and NCI-H460 were grown in RPMI-1640 culture medium (Roswell Park Memorial Institute, Biochrom, Cambridge, UK), supplemented with 5% heat-inactivated FBS (fetal bovine serum, Biochrom) and 1% of penicillin/streptomycin (Sigma-Aldrich, Lisboa, Portugal). HCT-15 cells were also cultured in the same medium but supplemented with 10% FBS. HPAEpic cells were cultivated in Dulbecco’s Modified Eagle Medium (DMEM, Lonza, Walkersville, MO, USA), supplemented with 10% FBS, 1% Non-Essential Amino Acids (NEAAs, Sigma-Aldrich, Lisboa, Portugal), and 1% penicillin/streptomycin.
Non essential amino acids neaa
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom
Non-essential amino acids (NEAA) are a group of amino acids that can be synthesized by the human body and are not required to be obtained from dietary sources. They play a vital role in various biological processes, including protein synthesis and cellular metabolism.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
L glutamine, by Thermo Fisher Scientific (5 mentions)
Penicillin, by Thermo Fisher Scientific (5 mentions)
Streptomycin, by Thermo Fisher Scientific (5 mentions)
Fetal bovine serum (fbs), by Merck Group (4 mentions)
Penicillin streptomycin, by Merck Group (3 mentions)
L glutamine, by Merck Group (3 mentions)
Sodium pyruvate, by Merck Group (3 mentions)
Rpmi 1640, by Merck Group (3 mentions)
Streptomycin, by Merck Group (3 mentions)
Dmem f12, by Merck Group (3 mentions)
Dulbecco s modified eagle s medium dmem, by Merck Group (3 mentions)
Glutamax, by Thermo Fisher Scientific (3 mentions)
Caco 2, by American Type Culture Collection (2 mentions)
Penicillin, by Merck Group (2 mentions)
β mercaptoethanol, by Merck Group (2 mentions)
Dulbecco s modified eagle medium dmem, by Lonza (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by GE Healthcare (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
30 protocols using non essential amino acids neaa
1
Diverse Tumor Cell Line Screening
2
Culturing Colon Fibroblasts CCD-18Co
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The cell line CCD-18Co (fibroblasts from the colon of a healthy human) was obtained from the American Type Cells Collections (Cat. No. ATCC-CRL-1459). The cells were cultured in Minimum Essential Medium Eagle (MEM) with Earle’s salts, L-glutamine, sodium bicarbonate, and phenol red, supplemented with 10% fetal bovine serum (FBS), 1% non-essential amino acids (NEAAs), and 1% penicillin/streptomycin solution (P/S), all purchased from Sigma-Aldrich (St. Louis, MO, USA). The incubation parameters were 37 ℃, 5% CO2, and 95% relative humidity.
3
A. platensis Extract Preparation and Use
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The A platensis was purchased from Martin Bauer GmbH (Vestenbergsgreuth, Germany). The water extract of both A platensis and phycocyanobilin was prepared as has been previously described elsewhere.4 The cell culture media and non‐essential amino acids (NEAAs) were obtained from Sigma‐Aldrich, and the other cell culture components were from Biosera (Nuaille, France). The serine/threonine phosphatase and protease inhibitor cocktails were purchased from either Sigma‐Aldrich or Serva. The Geltrex™ LDEV‐Free Reduced Growth Factor Basement Membrane Matrix was purchased from Thermo Fisher Scientific. The recombinant growth factors and inhibitors were procured as follows: rVEGF, rEGF (epidermal growth factor), rAREG (amphiregulin, autocrine mitogen related to EGF), rHGF/SF (hepatocyte growth factor/scatter factor), PD 0325901 (all from Sigma‐Aldrich), erlotinib (Cell Signaling Technology), vatalanib and axitinib (Selleck Chemicals) and bevacizumab (LGM Pharma). Unless otherwise specified, all other common chemicals were from Sigma‐Aldrich.
4
Culturing Human Colorectal Adenocarcinoma Cells
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Human colorectal adenocarcinoma cells (Caco-2) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained at 37 °C in a humidified atmosphere with 95% air and 5% CO2, and they were periodically screened for contamination. Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM)—high glucose (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% Fetal Bovine Serum (FBS, Sigma-Aldrich, St. Louis, MO, USA), 1% L-glutamine (Sigma-Aldrich, St. Louis, MO, USA), 100 U/mL penicillin/streptomycin (Sigma-Aldrich, St. Louis, MO, USA), 1% Non-Essential Amino Acids (NEAA) (Sigma-Aldrich, St. Louis, MO, USA), and 1% sodium pyruvate (Sigma-Aldrich, St. Louis, MO, USA).
5
Assessing Xanthohumol's Protective Effects
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Xanthohumol (XN), Aflatoxin B1 (AFB1), dimethylsulphoxide (DMSO), Benzo(a)pyrene (BaP), Minimal Essential Medium Eagle (MEM), NaHCO3, non-essential amino acids (NEAA), sodium pyruvate ethylenediaminetetraacetic acid (EDTA), NaCl, NaOH, and phenazine methosulfate (PMS) were all purchased from Sigma-Aldrich (St. Louis, MO, USA). Penicillin/streptomycin, phosphate-buffered saline (PBS), ethanol, fetal bovine serum, and L-glutamine were from PAA Laboratories (Toronto, Canada). Triton X-100 was from Thermo Fisher Scientific (Pittsburgh, PA, USA). Hoechst 33258, trypsin, low melting point agarose (LMP), and normal melting point agarose (NMP) were from Invitrogen (Waltham, MA, USA). The CellTiter96® AQueous cell proliferation assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MTS) was from Promega (Madison, WI, USA). Etoposide (ET) was from Santa Cruz Biotechnology (Dallas, TX, USA). GelRed Nucleic Acid Stain was from Biotium, (Fremont, CA, USA). Tris was from Merck (Darmstadt, Germany). Anti-γH2AX pS139, FITC conjugate human recombinant antibodies were from Miltenyi Biotec GmbH (Bergisch Gladbach, Germany). All other reagents were of the purest grade and solutions were made using Milli-Q water. Stock solutions of XN (70 mM) and AFB1 (3.2 mM) for the in vitro studies were prepared in DMSO and stored at −20 °C.
6
HPLC Analysis of Isoflavone Standards
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Dimethyl sulfoxide (DMSO), chloroform, HPLC grade acetonitrile, water, and formic acid were purchased from Sigma-Aldrich (Seelze, Germany). Reference standards for HPLC analysis of isoflavones: biochanin A, formononetin, genistein, genistein, glycitein, daidzein, ononin, were purchased from Fluka Chemie (Buchs, Switzerland). Methanol was from Avantor Performance Materials Poland S.A. (Gliwice, Poland). All reagents were of analytical grade. Distilled water was purchased from Sigma-Aldrich (Seelze, Germany). Cell culture media and supplements: DMEM/F12, DMEM low glucose, RPMI1640, MEM, non-essential amino acids (NEAA), sodium pyruvate, epidermal growth factor (EGF), insulin, hydrocortisone, cholera toxin, fetal bovine serum (FBS), donor horse serum, antibiotics solution (10,000 U penicillin and 10 mg streptomycin), phosphate buffered saline (PBS), trypsin solution, were purchased from Sigma-Aldrich (Seelze, Germany).
7
Ovine Meniscal Fibrocartilage Harvesting
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Natural Fibrocartilage cylinders (5.0 mm in diameter and 3.0 mm thick) were harvested from the avascular (white zone) of ovine menisci using a dermal biopsy punch. They were rinsed and incubated with phosphate buffered saline (Invitrogen Ltd, Paisley, UK, www.thermofisher.com ) containing 10% (v/v) Penicillin G (10.000 units per ml)/Streptomycin (10.000 μg/ml) antibiotic mixture (P/S; Sigma) and 1% (v/v) Amphotericin B (250 µg/ml; Sigma) for 20 minutes. Viability of the fibrocartilage disks was maintained by culture in basic medium containing Dulbecco's modified Eagle's medium (DMEM, Sigma) with 10 mM Hepes buffer (Sigma), P/S, 1% (v/v) nonessential amino acids (NEAA; Sigma), 1% (v/v) Glutamax (Sigma), and 10% Amphotericin B (Sigma) at 37°C in a 5% CO2 environment. The explants were used in the integration experiments within 3 days of culture.
8
Generation of iPSCs from LQTS3/BrS Fibroblasts
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Dermal fibroblasts obtained by dermal biopsy from a patient with LQTS3/BrS and two healthy volunteers were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich, St Louis, MO, USA) with 10% fetal bovine serum (FBS; Nichirei Biosciences, Tokyo, Japan), passaged twice, and used to generate iPSCs, as described below. Human iPSCs were maintained on irradiated mouse embryonic fibroblast (MEF) feeder cells in hiPSC culture medium, consisting of 80% DMEM/F12 (Sigma-Aldrich), 20% KO Serum Replacement (Invitrogen, Carlsbad, CA, USA), 4 ng/mL basic fibroblast growth factor (bFGF; WAKO, Osaka, Japan), 2 mmol/L l -glutamine (Invitrogen), 0.1 mmol/L non-essential amino acids (Sigma-Aldrich), 0.1 mmol/L 2-mercaptoethanol, 100 U/mL penicillin, and 100 μg/mL streptomycin (Invitrogen). The hiPSC medium was changed every 2 days and the cells were passaged using 1 mg/mL collagenase IV (Invitrogen) every 5–7 days. 293FT cells were cultured in DMEM supplemented with 10% FBS (Nichirei Biosciences), 1 × 10−4 M non-essential amino acids (NEAA; Sigma-Aldrich), 2 mmol/L l -glutamine (Invitrogen), 100 U/mL penicillin, and 100 μg/mL streptomycin (Invitrogen).
9
Evaluation of Antioxidant and Antimicrobial Potential
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Folin-Ciocalteu reagent, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), sodium carbonate (Na2CO3), 2,2-diphenyl-1-picrylhydrazyl (DPPH), caffeic acid, luteolin, hydrogen peroxide (H2O2), Thiazolyl Blue Tetrazolium Bromide (MTT), Trypic Soy Broth, Sabouraud Dextrose Broth, Mueller Hinton Broth and RPMI. Eagle’s Minimum Essential Medium (MEM), fetal bovine serum (FBS), RPMI-1640 medium, Dulbecco’s modified Eagle’s medium (DMEM), Non-essential Amino Acids (NEAA), and all HPLC and GC standard markers were obtained from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals used were of analytical grade and water was ultra-pure.
10
Biopolymer Synthesis and Characterization
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1-Ethyl-3-methylimidazolium
acetate ([EMIm][OAc]),
dimethyl sulfoxide (DMSO), chitosan (low MW and medium MW), plant
α-cellulose, acetic acid, glucose, yeast extract, peptone, anhydrous
disodium phosphate, citric acid monohydrate, acetate buffer, sodium
acetate, ninhydrin, hydrindantin, 2-methoxyethanol, phosphate-buffered
saline (PBS), polystyrene latex particles, Dulbecco’s modified
Eagle’s medium (DMEM), fetal bovine serum (FBS), sodium pyruvate,
nonessential amino acids (NEAA), penicillin streptomycin (pen strep),
formalin, and methanol (MeOH) were purchased from Sigma-Aldrich. Fluorescein
phalloidin (FITC), and 4′,6-diamidino-2-phenylindole (DAPI)
were purchased from Thermo Fisher Scientific. Plant α-cellulose
and [EMIm][OAc] were dried at 60 °C en vacuo overnight; and DMSO
dried over activated 4 Å molecular sieves before use.
acetate ([EMIm][OAc]),
dimethyl sulfoxide (DMSO), chitosan (low MW and medium MW), plant
α-cellulose, acetic acid, glucose, yeast extract, peptone, anhydrous
disodium phosphate, citric acid monohydrate, acetate buffer, sodium
acetate, ninhydrin, hydrindantin, 2-methoxyethanol, phosphate-buffered
saline (PBS), polystyrene latex particles, Dulbecco’s modified
Eagle’s medium (DMEM), fetal bovine serum (FBS), sodium pyruvate,
nonessential amino acids (NEAA), penicillin streptomycin (pen strep),
formalin, and methanol (MeOH) were purchased from Sigma-Aldrich. Fluorescein
phalloidin (FITC), and 4′,6-diamidino-2-phenylindole (DAPI)
were purchased from Thermo Fisher Scientific. Plant α-cellulose
and [EMIm][OAc] were dried at 60 °C en vacuo overnight; and DMSO
dried over activated 4 Å molecular sieves before use.
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