N 2 hydroxyethylpiperazine n 2 ethane sulfonic acid

Manufactured by Thermo Fisher Scientific

Sourced in United States

N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid is a chemical compound commonly used as a buffering agent in various laboratory applications. It is a zwitterionic organic compound that helps maintain a stable pH environment in aqueous solutions.

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11 protocols using n 2 hydroxyethylpiperazine n 2 ethane sulfonic acid

Adipogenesis Assay in 3T3-L1 Cells

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All chemicals were of the highest quality available (e.g., analytical grade). Bovine calf serum and 3T3-L1 mouse embryonic fibroblasts and were purchased from the American Type Culture Collection (ATCC, USA). Dulbecco's modified Eagle's medium (DMEM), penicillin, streptomycin, fetal bovine serum (FBS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-nyltetrazolium bromide (MTT), and N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid were obtained from Gibco Invitrogen (Grand Island, NY, USA). Insulin solution (bovine), 3-isobutyl-1-methylxanthine (IBMX), 4-nitrophenyl dodecanoate (pNP laurate), porcine pancreas lipase, and simvastatin (SIM) were obtained from Sigma-Aldrich (St. Louis, USA). Dimethyl sulfoxide (DMSO) was purchased from Carlo Erba Reagents S.r.l. (Chaussée du Vexin, Val de Reuil, USA). Dexamethasone was obtained from G Bioscience (St. Louis, USA). Oil Red O was purchased from Amresco Inc. (Solon, OH, USA).

Tiamyom K., Sirichaiwetchakoon K., Hengpratom T., Kupittayanant S., Srisawat R., Thaeomor A, & Eumkeb G. (2019). The Effects of Cordyceps sinensis (Berk.) Sacc. and Gymnema inodorum (Lour.) Decne. Extracts on Adipogenesis and Lipase Activity In Vitro. Evidence-based Complementary and Alternative Medicine : eCAM, 2019, 5370473.

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Isolation and Induction of Naive and Regulatory T Cells

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Naive CD4+ T cells were isolated by an EasySep™ Mouse Naïve CD4+ T Cell Isolation kit (STEMCELL Technologies, Canada). CD4+CD25+ nTregs sorted from the thymus of C57BL/6 mice were expanded with anti-mouse CD3/CD28 beads (1:3) and IL-2 (200 IU/ml) for 7 days and were then harvested. For iTreg induction, naive CD4+ T cells (3×10⁵ cells/well) were stimulated with anti-mouse CD3/CD28 beads (1:5, Miltenyi Biotec) in the presence of rhIL-2 (50 IU/ml, BioLegend), all-trans retinoic acid (atRA) (5 nM) and 100 μg/ml vitamin C with rhTGF-β (5 ng/ml, BioLegend) in 96-well plates for 7 days as previously described (6 (link)). RPMI 1640 medium (Gibco) was supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin, 10 mM N-2-Hydroxyethylpiperazine-N’−2’-ethanesulfonic Acid (Invitrogen Life Technologies), 1×10⁵ M 2-mercaptoethanol (Sigma-Aldrich), and 10% heat-inactivated FBS (Hy Clone) and was used for all cultures. Foxp3 expression was evaluated using a Fortessa (BD Biosciences).

Su W., Chen X., Zhu W., Yu J., Li W., Li Y., Li Z., Olsen N., Liang D, & Zheng S.G. (2019). cAMP-Adenosine Feedback Loop Maintains the Suppressive Function of Regulatory T Cells. Journal of immunology (Baltimore, Md. : 1950), 203(6), 1436-1446.

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Culturing MDCK and HEK293T Cells

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Madin-Darby canine kidney (MDCK) cells and human embryonic kidney (HEK) 293T cells purchased from American type cell culture (ATCC) were grown in minimum essential media (MEM) and Opti-MEM, respectively (Gibco). The MEM used for cell culture was supplemented with 10% fetal bovine serum (Gibco), penicillin (100 U/mL) and streptomycin (100 μg/mL) (Gibco), vitamins (Sigma-Aldrich), and N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES, Gibco). The infection media (MEM, Gibco) used for viral infection was supplemented with 0.3% bovine serum albumin (Sigma-Aldrich), penicillin (100 U/mL) and streptomycin (100 μg/mL) (Gibco), vitamins (Sigma-Aldrich) and HEPES (Gibco).

Daulagala P., Mann B.R., Leung K., Lau E.H., Yung L., Lei R., Nizami S.I., Wu J.T., Chiu S.S., Daniels R.S., Wu N.C., Wentworth D., Peiris M, & Yen H.L. (2023). Imprinted Anti-Hemagglutinin and Anti-Neuraminidase Antibody Responses after Childhood Infections of A(H1N1) and A(H1N1)pdm09 Influenza Viruses. mBio, 14(3), e00084-23.

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SARS-CoV-2 Variant Propagation in Vero.E6 Cells

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Vero.E6 cells (ATCC CRL-1586, clone E6) were kept in culture using Dulbecco’s modified Eagle medium (Gibco, Waltham, MA), which was supplemented with 10 mL of Antibiotic-Antimycotic (100 U/ml penicillin–100 μg/ml streptomycin–0.25 μg/ml amphotericin B; Gibco), 10% of fetal bovine serum (FBS; Corning, Corning, NY), and 1% HEPES (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid; Gibco). Wild-type SARS-CoV-2 (isolate USA-WA1/2020), hCoV-19/South Africa/KRISP-K005325/2020 (B.1.351, BEI Resources NR-54009), hCoV-19/Japan/TY7–503/2021 (P.1, BEI resources NR-54982), and hCoV-19/England/204820464/2020 (B.1.1.7, BEI Resources NR54000) were cultured in Vero.E6 cells for 3 days at 37°C and then the supernatant was clarified via centrifugation at 1,000g for 10 minutes. Virus stocks were stored at −80°C. The protocol was described in greater detail previously [22 (link),39 (link)]. All work with authentic SARS-CoV-2 was performed in a biosafety level 3 (BSL-3) facility following institutional guidelines.

Amanat F., Strohmeier S., Meade P.S., Dambrauskas N., Mühlemann B., Smith D.J., Vigdorovich V., Sather D.N., Coughlan L, & Krammer F. (2021). Vaccination with SARS-CoV-2 variants of concern protects mice from challenge with wild-type virus. PLoS Biology, 19(12), e3001384.

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Wound Tissue Dissociation for scRNA-seq

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Wound beds were digested for scRNA-seq analysis in a buffer of Roswell Park Memorial Institute (RPMI) medium with glutamine (Gibco), Liberase Thermolysin Medium (TM) (Roche), DNase, N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (Gibco), sodium pyruvate (Gibco), non-essential amino acids (Gibco), and antibiotic-antimycotic (100X) (Gibco). Blood cells were removed with ammonium-chloride-potassium (ACK) lysing buffer (Gibco). Cells were resuspended in Dulbecco’s Modified Eagle’s Medium (DMEM) (ATCC) with 0.1% bovine serum albumin (BSA) for analysis.

Justynski O., Bridges K., Krause W., Forni M.F., Phan Q.M., Sandoval-Schaefer T., Carter K., King D.E., Hsia H.C., Gazes M.I., Vyce S.D., Driskell R.R., Miller-Jensen K, & Horsley V. (2023). Apoptosis recognition receptors regulate skin tissue repair in mice. eLife, 12, e86269.

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Isolation and Culture of Blood Cells

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Blood outgrowth endothelial cells (BOECs) were obtained from healthy controls and cultured in Endothelial Basal Medium 2 (Lonza, Walkersville, MD) supplemented with Endothelial Cell Growth Medium (EGM-2 BulletKit; Lonza Walkersville Inc., Walkersville, MD), 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO), and 5% antibiotic-antimycotic mix (Sigma).¹³ (link) Neutrophils were isolated from whole blood as described.¹⁴ (link) In short, neutrophils were isolated using Polymorphprep (Axis-Shield, Dundee, Scotland), erythrocytes were lysed, and washed neutrophils were resuspended in Roswell Park Memorial Institute 1640 medium (WISENT, St Bruno, QC, Canada) containing 10 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (Gibco, Life Technologies, Carlsbad, CA). Platelets were isolated from same donors as described.¹⁵ (link) In short, whole blood was drawn in acid citrate dextrose (1:7) tubes and after centrifugation platelet-rich plasma was isolated and washed twice with phosphate-buffered saline/acid citrate dextrose.

Riedl M., Noone D.G., Khan M.A., Pluthero F.G., Kahr W.H., Palaniyar N, & Licht C. (2016). Complement Activation Induces Neutrophil Adhesion and Neutrophil-Platelet Aggregate Formation on Vascular Endothelial Cells. Kidney International Reports, 2(1), 66-75.

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Culturing Human Cell Lines Huh-7 and 293T

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Huh-7 (human, liver, RRID: CVCL_0336) and 293T (human, kidney, RRID: CVCL_0063) cell lines were obtained from the Japan Research Biological Resources Collection and American Type Culture Collection, respectively. The cell line has been checked by the cell identification assay and is free from other cellular contamination. In addition, exogenous virus factor test was also performed, showing no exogenous virus contamination. Sterility testing and mycoplasma testing have been performed recently, and all meet the requirements. All cell lines were cultured in Dulbecco's modified Eagle's medium (HyClone, Logan, UT) with 100 U/ml of penicillin-streptomycin solution (GIBCO, Grand Island, NY), 20 mM N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (GIBCO), and 10% fetal bovine serum (PAN-Biotech, Adenbach, Germany) at 37 °C in a humidified atmosphere with 5% CO₂.

Zhang M., An Y., Wu X., Cai M., Zhang X., Yang C., Tong J., Cui Z., Li X., Huang W., Zhao C, & Wang Y. (2022). Retrospective immunogenicity analysis of seasonal flu H3N2 vaccines recommended in the past ten years using immunized animal sera. eBioMedicine, 86, 104350.

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Culturing HEK 293T Cell Lines

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The HEK 293T cell lines (CRL-3216) were obtained from the American Type Culture Collection. It was cultured in Dulbecco's modified Eagle's medium (HyClone, Logan, UT) with 100 U/mL of penicillin-streptomycin solution (GIBCO, Grand Island, NY), 20 mM N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (GIBCO), and 10% fetal bovine serum (PAN-Biotech, Aidenbach, Germany) at 37 , 5% CO 2 environment.

Cai M., Hu Z., Yang Y., Mao T., Liu Y., Lu G., Yang F., Qi J., Huang W, & Wang Y. (2023). Characterization of a human-mouse chimeric monoclonal antibody targeting rabies virus glycoprotein. Journal of medical virology, 95(7).

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Cultivation and Infection of Vero.E6 Cells with SARS-CoV-2

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Vero.E6 cells (ATCC CRL-1586, clone E6) were maintained for cell culture using Dulbecco’s modified Eagle medium (Gibco), which was supplemented with Antibiotic-Antimycotic (100 U/mL penicillin, 100 μg/mL streptomycin, 0.25 μg/mL amphotericin B; Gibco), 10% of fetal bovine serum (FBS; Corning), and 1% HEPES (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid; Gibco). Wild-type SARS-CoV-2 (isolate USA-WA1/2020) was grown in Vero.E6 cells for 3 days at 37°C and then the supernatant was clarified via centrifugation at 1,000 g for 10 min. Virus stocks were stored at −80°C. The protocol is described in greater detail previously (44 (link), 45 (link)). All work with authentic live SARS-CoV-2 was performed in the biosafety level 3 (BSL-3) facility following institutional guidelines.

Amanat F., Clark J., Carreño J.M., Strohmeier S., Yellin T., Meade P.S., Bhavsar D., Muramatsu H., Sun W., Coughlan L., Pardi N, & Krammer F. (2023). Immunity to Seasonal Coronavirus Spike Proteins Does Not Protect from SARS-CoV-2 Challenge in a Mouse Model but Has No Detrimental Effect on Protection Mediated by COVID-19 mRNA Vaccination. Journal of Virology, 97(3), e01664-22.

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Murine and Human Breast Cancer Cell Culture

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Murine EMT-6 (gift from Dr. David Murray, Department of Oncology, University of Alberta, Edmonton, AB, Canada), human MCF-7 and human MDA-MB-231 breast cancer cell lines (American Type Culture Collection, Manassas, VA, USA) were grown in a CO₂ incubator at 37 °C, in Gibco Dulbecco’s modified Eagle’s medium/Nutrient Mixture F-12 (Life Technologies) supplemented with 15 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (Gibco catalog number 15630; Life Technologies), 2 mM l-glutamine, 10 % fetal bovine serum (FBS, Gibco catalog number 12483; Life Technologies) and 1 % penicillin-streptomycin in T75 flasks with media renewed every 2–3 days.

Wuest M., Kuchar M., Sharma S.K., Richter S., Hamann I., Wang M., Vos L., Mackey J.R., Wuest F, & Löser R. (2015). Targeting lysyl oxidase for molecular imaging in breast cancer. Breast Cancer Research : BCR, 17(1), 107.

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