Cell isolation from the lungs was performed as previously described (37 (link),38 (link)). In brief, the right lung was excised after the ligation of the left main bronchus. The whole lungs were minced, homogenized and subsequently incubated with shaking for 30 min at 37°C with the collagenase type IV solution (cat. no. C8160; Beijing Solarbio Science & Technology Co., Ltd.). The supernatant was removed and then filtered through a nylon mesh with pore size 48 µm (cat. no. YA0691; Beijing Solarbio Science & Technology Co., Ltd.), and the resulting cells were washed in complete medium [CM; RPMI-1640 + 10% heat-inactivated fetal bovine serum + 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer + 10 mM non-essential amino acids + 10 mM sodium pyruvate + 10 U/ml penicillin/streptomycin; all from Sigma-Aldrich]. Subsequently, the isolated cells were enriched by density gradient centrifugation at 800 × g for 30 min at 4°C (Percoll, Sigma-Aldrich; Merck KGaA) and they were re-suspended in CM for further analysis.
4 2 hydroxyethyl 1 piperazineethanesulfonic acid hepes buffer
Manufactured by Merck Group
Sourced in United States
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) is a chemical compound used as a buffer in biological research and cell culture applications. It is a zwitterionic organic compound that helps maintain a stable pH environment in aqueous solutions.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by Merck Group (2 mentions)
Streptomycin, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Non essential amino acids (neaa), by Merck Group (1 mentions)
Percoll, by Merck Group (1 mentions)
Rpmi 1640, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Lead citrate, by Merck Group (1 mentions)
Uranyl acetate, by Merck Group (1 mentions)
Atp determination kit, by Thermo Fisher Scientific (1 mentions)
Osmium tetroxide, by Merck Group (1 mentions)
Trypsin edta solution, by HiMedia (1 mentions)
Epoxy embedding medium kit, by Merck Group (1 mentions)
Mtt dye, by HiMedia (1 mentions)
Trypan blue, by HiMedia (1 mentions)
Fluorescein isothiocyanate phalloidin fitc phalloidin, by Thermo Fisher Scientific (1 mentions)
Sod assay kit, by Merck Group (1 mentions)
Rotenone, by Merck Group (1 mentions)
8 protocols using 4 2 hydroxyethyl 1 piperazineethanesulfonic acid hepes buffer
1
Isolation of Lung Cells for Analysis
2
Comprehensive Cell Culture Protocols
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Chemicals such as Dulbecco’s Modified Eagle’s Medium (DMEM), l -Glutamine, Fetal Bovine Serum (FBS), Trypsin-EDTA solution, Antibiotic-Antimycotic solution (10000 U/mL Penicillin, 10 mg/mL Streptomycin and 25 μg/mL Amphotericin B in 0.9 % normal saline for 100 X), MTT dye, Dimethyl Sulfoxide (DMSO), Trypan blue, Dulbecco’s Phosphate Buffered Saline (DPBS), Tris-EDTA, Propidium iodide (PI), Ribonuclease A (RNase A) and Triton X-100 were purchased from Himedia, India. Molecular probes, Fluorescein isothiocyanate-Phalloidin (FITC-Phalloidin), and 4′,6- diamidino-2-phenylindole (DAPI) were obtained from ThermoFisher Scientific, USA. DCFDA, 3,3′-dihexyloxacarcocyanine iodide (DiOC6), Rotenone, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, disodium phosphate (Na2HPO4) 25 % (v/v) glutaraldehyde, paraformaldehyde, osmium tetroxide, uranyl acetate, and lead citrate were purchased from Sigma Aldrich, USA. The kits used in this study such as the SOD Assay kit and Epoxy Embedding Medium Kit were procured from Sigma Aldrich, USA (Cat. No.:19106 and Cat. No.: 45359-1EA-F respectively), and the ATP Determination kit was purchased from Thermo Scientific, USA (Cat. No.: A22066). The absolute ethanol used in this study is of HPLC grade (Commercial Alcohols, Greenfield Global, Canada). All the reagents and chemicals are of analytical grade.
3
Fucoxanthin Extraction and Anti-Inflammatory Evaluation
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The fucoxanthin was extracted from brown algae Laminaria japonica. Fucoxanthin extract was provided by Oryza Oil & Fat Chemical Co., Ltd. (Tokyo, Japan). The RAW 264.7 macrophage cells were supplied from American Type Culture Collection (ATCC). Dulbecco’s Modified Eagle Medium (DMEM) was purchased from GIBCO (Carlsbad, CA, USA). The fetal calf serum (FCS) was purchased from HyClone (Carlsbad, CA, USA). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) powder, nitro blue tetrazolium (NBT) powder, dimethyl sulfoxide (DMSO), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, dichloro-dihydro-fluorescein diacetate (DCFH-DA), and Trypan Blue were purchased from Sigma Aldrich (St. Louis, MO, USA). The glucose enzymatic kit was purchased from Kyokuto Pharmaceutical Industrial Co., Ltd. (Tokyo, Japan) and insulin ELISA kits was purchased from Mercodia AB Inc. (Uppsala, Sweden). Luteinizing hormone (LH) RIA kits were provided by Dr. A. F. Parlow, National Institute of Diabetes and Digestive and Kidney Diseases, National Hormone and Peptide Program (Torrance, CA, USA) and testosterone EIA as well as enzymatic antioxidant (catalase, glutathione peroxidase, superoxide dismutase) commercial kits were purchased from Eugene Chen Co., Ltd. (Taipei, Taiwan).
4
Caco-2 Cell Culture Methodology
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The human colon epithelial cell line Caco-2 (ATCC HTB-37™, LGC Standards, Łomianki, Poland) was used in the experiment. Caco-2 cells were propagated in a complete growth medium (CGM), i.e., Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma–Aldrich, Poznań, Poland) containing 4500 mg/L of glucose. The medium was supplemented with a 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Warsaw, Poland), a 2% 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (Sigma–Aldrich) and a 1% solution of minimum essential medium (MEM) non-essential amino acid (Thermo Fisher Scientific, Warsaw, Poland). Furthermore, 1% of the medium constituted antibiotics (gentamycin, penicillin, streptomycin and amphotericin B (Sigma-Aldrich, Poznań, Poland). After reaching 80% confluence, cultures were passaged using a trypsin– ethylenediaminetetraacetic acid (EDTA) solution (TrypLE, Thermo Fisher Scientific, Warsaw, Poland).
5
Reagents for Cell Culture Experiments
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Dulbecco’s modified Eagle medium (DMEM), penicillin/streptomycin (P/S), L-glutamine (L-Gln), and foetal bovine serum (FBS) were purchased from Lonza (Barcelona, Spain). Non-essential amino acids (NEAA), amphotericin B, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, Palm, non-essential fatty acid (NEFA), free bovine serum albumin (BSA), 2-mercaptoethanol, and CaCl2 were obtained from Sigma Aldrich (Madrid, Spain). Glc, neutral red dye, and glacial acetic acid were purchased from Panreac AppliChem (Barcelona, Spain). RSV was purchased from Fagron Iberica (Barcelona, Spain) and resveratrol-3-sulfate was obtained from Bertin Pharma (Montigny le Bretonneux, France). Formaldehyde and ethanol were obtained from Millipore (Madrid, Spain). All LC-MS-grade solvents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Finally, recombinant human leptin was obtained from BioVision (San Francisco, CA, USA).
6
Electrochemical Detection of CEA
7
In Vitro Evaluation of Anti-Cancer Potential
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In this study, we work on human MCF7 breast cancer cells (ATCC® HTB-22), human PC3 prostate cancer cell line (ATCC® CRL-1435), A375 human skin cancer cell line (ATCC® CRL-1619), PANC1 pancreatic cell line (ATCC® CRL-1469), A549 lung cancer cell line (ATCC® CCL-185) and colorectal cancer cell line CACO2 (ATCC HTB-37).[33 (link)] All of the cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM) high glucose containing 10% Fetal Bovine Serum (FBS) (Bio Whittaker, Verviers, Belgium), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) Buffer (10 mM), gentamicin (50 μg/mL), L-glutamine (100 μg/mL), streptomycin (100 μg/mL), penicillin (100 μg/mL), (Sigma, St. Luis, MO, USA). Trypsin-EDTA (Biowest, USA) was routinely used for subcultures. Cell growth was accomplished at 37°C in a 5% carbon dioxide 95% air atmosphere in CO2 incubator (EuroClone, Italy). Sulforhodamine B[33 (link)] was from Santa Cruz Biotechnology, Inc. Texas (USA). 3-(4,5-dimethylthiazol-2-yl)−2,5- diphenyltetrazolium bromide (MTT) was from (Sigma, USA).
8
Detailed Labeling Protocols for NMR Studies
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All 13C labeled or unlabeled chemicals utilized in this study were purchased from commercial sources and used without further purification. [U-13C6] glucose and [U-13C2] acetate were purchased from Sigma Aldrich (St. Louis, MO). [U-13C3] pyruvate along with deuterated 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) and deuterium oxide (D2O) were obtained from Cambridge Isotope Laboratories (Tewksbury, MA). Unlabeled buffer agents (mono-and bi-basic phosphates, sodium azide, sodium chloride (NaCl), potassium chloride (KCl), calcium chloride (CaCl2), magnesium sulfate (MgSO4), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, unlabeled glucose, and ethylene diamine tetra-acetic acid (EDTA)) were also obtained from Sigma Aldrich, St Louis, MO, USA. Ringer’s solution ((in mM) 120 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2KH2PO4, 1.2MgSO4, 25 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, and 5.5 glucose) gas equilibrated with 95% O2 and 5% CO2 to pH 7.4 or minimum essential media (MEM) (Fisher) containing 292 mM L-glutamine, 52 mM L-isoleucine, 52 mM L-leucine, 15 mM L-methionine, 32 mM L-phenylalanine, 48 mM L-threonine, 10 mM L-tryptophan, 52 mM L-Tyrosine disodium salt dehydrate, and 46 mM valine, was used to support isolated muscles.
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