Following storage at −140 °C for 2 wk, isolated cells and microbeads were rapidly thawed in a 37 °C water bath24 (link),28 (link) using ice-cold Eagle’s minimum essential medium (EMEM) (EMEM; Lonza, Verviers, Belgium) as thawing media. Similarly, cryopreserved HMBs were thawed with 2% (v/v) HSA added in EMEM. Thawed hepatocytes were then plated and cultured in William’s E medium (WEM; Sigma-Aldrich) supplemented with 10% FCS (Invitrogen, Paisley, UK), 10-mM 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (HEPES; Lonza), 2-mM l -glutamine (Sigma-Aldrich), 0.1-μM dexamethasone (Sigma-Aldrich), 0.1-μM insulin (Sigma-Aldrich), penicillin (50 U/mL; Sigma-Aldrich), and streptomycin (50 μg/mL; Sigma-Aldrich). Thawed microbeads were cultured in the same medium as the same as hepatocytes except Connaught Medical Research Laboratories medium (Mediatech was used, Inc., Vancouver, Canada) instead of WEM without HEPES. The cells and microbeads were maintained in a humidified incubator at 37 °C, 5% CO2 for 7 d.
4 2 hydroxyethyl 1 piperazineethanesulfonic acid hepes
Manufactured by Lonza
Sourced in United Kingdom, Switzerland, Germany, United States, Belgium
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) is a chemical compound used as a buffering agent in biological research and industry. It maintains a stable pH in aqueous solutions, primarily in the range of 6.8 to 8.2. HEPES is commonly used in cell culture media, enzyme assays, and other biochemical applications where pH control is critical.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Insulin, by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Osmium tetroxide, by Agar Scientific (1 mentions)
Milli q system, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Dutscher (1 mentions)
Gentamycin, by Lonza (1 mentions)
Rpmi 1640 glutamax medium, by Thermo Fisher Scientific (1 mentions)
Rhil 2, by Thermo Fisher Scientific (1 mentions)
Rh il4, by Miltenyi Biotec (1 mentions)
Phytohemagglutinin m pha m, by Merck Group (1 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
Human ab serum, by Merck Group (1 mentions)
Thrombin, by Merck Group (1 mentions)
Alexa 647 conjugated fibrinogen, by Thermo Fisher Scientific (1 mentions)
Factor xiiia, by CSL Behring (1 mentions)
7 protocols using 4 2 hydroxyethyl 1 piperazineethanesulfonic acid hepes
1
Cryopreserved Cell Thawing and Culture
2
Antioxidant Enzyme Activity Analysis
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If not otherwise stated, chemicals were obtained from Sigma-Aldrich (St Louis, MO, USA). Phenol-red free Dulbecco’s Modified Eagle’s Medium (DMEM) with 4.5 g/L glucose and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) as the buffering agent (product number 12-709) was obtained from Lonza (Basel, Switzerland). The plastic and glassware used for chemical analysis were from Sarstedt (Nümbrecht, Germany). Osmium tetroxide was purchased from Agar Scientific (Stansted, UK) and epoxy resin (medium hard) from TAAB Laboratories Equipment (Aldermaston, UK). GPx and SOD assay kits were purchased from Cayman Chemical (Ann Arbor, MI, USA). All dilutions were made with high purity deionized water (18.2 MΩcm), obtained from a Milli-Q® system (EMD Millipore, Billerica, MA, USA).
3
Cell Culture of MRL/N-1 Cells
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MRL/N-1 cells were obtained from Tetsuya Kodama, Tohoku University School of Medicine, Japan. They were routinely cultured in Roswell Park Memorial Institute medium (RPMI) 1640 buffered-medium supplemented with 10% (v/v) fetal bovine serum (Dutscher), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and gentamycin (10 µg/ml; Lonza BioWhittaker). The absence of mycoplasma contamination was systematically ensured.
4
In vitro Expansion of Human ILC2s
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Sorted ILC2s (100 cells/well) were expanded with human feeder PBMCs (100,000/well; 37 °C, 5% CO2) for three to five weeks. Culture medium contained RPMI 1640 Glutamax medium (Life Technologies, Darmstadt, Germany), 1% Pen/Strep (Life Technologies, Darmstadt, Germany), 10% h.i. human AB serum (Sigma-Aldrich, Taufkirchen, Germany), and 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid ( HEPES, Lonza, Wakersville, USA). The medium was supplemented with 100 U/ml rh-IL-2 (Life Technologies, Darmstadt, Germany), 25 ng/ml rh-IL-4 (Miltenyi Biotech, Bergisch Gladbach, Germany), 5 µg/ml phytohemagglutinin-M (PHA-M; Sigma-Aldrich, Taufkirchen, Germany).
5
Engineered Fibrin Matrix Preparation
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Fibrin matrices were prepared by mixing human fibrinogen (plasminogen-, von Willebrand Factor-, and fibronectin-depleted; 25 mg/mL; Enzymes Research Laboratories, Indiana, USA), fluorescent Alexa 647-conjugated fibrinogen (0.5 mg/mL; Invitrogen, California, USA), factor XIIIa (3 U/mL; CSL Behring, Pennsylvania, USA), and thrombin (6 U/mL; Sigma-Aldrich, Missouri, USA) with 2.5 mM Ca2+ in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes) (Lonza, Basel, CH). Matrices containing TG-VEGF and TG-PDG-BB were obtained by adding the engineered proteins to the cross-linking enzymes solution before mixing with fibrinogen.
6
SARS-CoV-2 and Influenza A Virus Propagation
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SARS-CoV-2 isolate USA-WA1/2020 (NR-52281) (Biodefense and Emerging Infections Research Resources Repository, BEI Resources) was propagated in Vero-E6 cells in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco) supplemented with 2% fetal bovine serum (FBS) (Millipore Sigma), 1mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Lonza Bioscience) and 1% penicillin/streptomycin (Thermo Fisher Scientific). Virus stocks were filtered by centrifugation with Amicon Ultra-15 Centrifugal filter unit (Sigma-Aldrich) and sequenced to ensure maintenance of the furin cleavage site. Infectious viral titers were quantified by plaque assay in Vero-E6 cells (American Type Culture Collection, ATCC) in DMEM supplemented with 2% FBS, 1mM HEPES and 0.7% OXOID agar (Thermo Fisher Scientific). Assays were fixed in 5% paraformaldehyde (PFA) (Thermo Fisher Scientific) and stained with crystal violet (Sigma-Aldrich). All infections were performed with either passage 3 or 4 SARS-CoV-2. Influenza A virus H1N1 isolate A/California/04/2009 (BEI Resources) was propagated in Madin-Darby canine kidney (MDCK) (ATCC) cells in DMEM supplemented with 0.35% bovine serum albumin (BSA) (Thermo Fisher Scientific), filtered and sequenced in a manner comparable to SARS-CoV-2 stocks. All cells used in this study were routinely tested for the presence of mycoplasma using MycoAlert Mycoplasma Detection Kit (Lonza).
7
Chitosan Characterization and Drug Evaluation
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Chitosan hydrochloride was obtained from Heppe Medical GmbH, Halle, Germany. The following characteristics applied: DD 92.7%; viscosity 4-5 mPas at 1% in water at 20°C for TIM-1 studies and Ussing type rat model and DD 93.05%, viscosity 5.9 mPas at 1% in water at 20°C for Caco-2 and the inTESTine. Zovirax 200 mg dispersible tablets (GlaxoSmithKline, UK) were purchased in the Netherlands. Acyclovir was obtained from Fagron (Fagron, The Netherlands). For the Caco-2 studies, Hanks' balanced salt solution (HBSS), Dulbecco's modified Eagle's medium (DMEM), 10,000 IU/ml penicillin and 10,000 μg/ml streptomycin, nonessential amino acid medium (100 x) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) were obtained from Lonza (Verviers, Belgium). Fetal bovine serum (FBS) was purchased from Biological Industries (Beit Haemek, Israel). 2-(N-morpholino)ethanesulfonic acid (MES) was obtained from Sigma-Aldrich (St. Louis, MO, United States). For rat ligated loop studies, ketamin (Ketavet, Pfizer, Germany), and xylazin (Rompun, Bayer, Germany) were obtained via the Pharmacy of the Medical Center of the Johannes Gutenberg University, Mainz, Germany. For the InTESTine study, 14 C-Antipyrine (55 mCi/mmol) was purchased from American Radiolabeled Chemicals Inc. (St. Louis, Missouri, United States).
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