β mercaptoethanol

Manufactured by Carl Roth

Sourced in Germany

β-mercaptoethanol is a reducing agent commonly used in biochemical applications. It acts by breaking disulfide bonds in proteins, thereby maintaining their reduced state. This colorless, odorous liquid is soluble in water and organic solvents.

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22 protocols using β mercaptoethanol

Authentication and Characterization of Retinoblastoma Cell Lines

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The human RB cell line RB-355, established and first described by Griegel et al (1990) (8 (link)), and formerly donated by K. Heise, was kindly provided by Dr H. Stephan. The RB cell lines Y-79 (9 (link)) and WERI-Rb1 (10 (link)), originally purchased from the Leibniz Institute DSMZ (German Collection of Microorganisms and Cell Cultures), were likewise kindly provided by Dr H. Stephan. All RB cell lines were last tested and authenticated in September 2015. Mutation analyses were conducted using an MLPA kit (SALSA MLPA kit P047 RB1; MRC-Holland, Amsterdam, The Netherlands) and reactions were performed according to the manufacturer's instructions. Additional sequencing of the RB1 gene was performed for all RB cell lines. However, most recent STR analyses (March 2017) confirmed the authenticity of the cell lines.
The cell lines were cultivated as suspension cultures in Dulbecco's modified Eagle's medium (DMEM) with 15% fetal bovine serum (FBS) (both from PAN-Biotech GmbH, Aidenbach, Germany), 100 U penicillin/ml and 100 µg streptomycin/ml, 4 mM L-glutamine (both from Gibco, Karlsruhe, Germany), 50 µM β-mercaptoethanol (Carl Roth, Karlsruhe, Germany) and 10 µg insulin/ml (PAN-Biotech) at 37°C, 10% CO₂ and 95% humidity. No approval from an Ethics Committee was required for work with the human cell lines.

Busch M., Papior D., Stephan H, & Dünker N. (2017). Characterization of etoposide- and cisplatin-chemoresistant retinoblastoma cell lines. Oncology Reports, 39(1), 160-172.

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Proliferation Assay for T Cells

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Untouched CD4⁺ and CD8⁺ T cells derived from naïve mice or nylon wool-enriched T cells [17 (link)] were resuspended in medium (IMDM supplemented with 5% FCS (PAA, Cölbe, Germany), 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin (all from Sigma-Aldrich, Deisenhofen, Germany), and 50 μM β-mercaptoethanol (Carl Roth, Karlsruhe, Germany)) at a density of 5 × 10⁴/100 μl and were seeded on 96-well tissue-culture plates (Corning, Cambridge, MA). In some experiments, T cells were serially diluted in triplicates. T cell populations were polyclonally stimulated with plate-bound anti-CD3- (1 μg/ml) and anti-CD28- (2 μg/ml) specific antibodies (both from Affymetrix/eBioscience, San Diego, CA) for 96 h. In order to assess the proliferation of T cells, ³H thymidine (0.5 μCi/well) was applied for the last 16-18 h of the incubation time. Then, cells were harvested onto glass fiber filters, and retained radioactivity was measured in a liquid scintillation counter (1450 MicroBeta TriLux, LKB Wallac, Turku, Finland).

Käfer R., Schmidtke L., Schrick K., Montermann E., Bros M., Kleinert H, & Pautz A. (2019). The RNA-Binding Protein KSRP Modulates Cytokine Expression of CD4+ T Cells. Journal of Immunology Research, 2019, 4726532.

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Eμ-Myc Lymphoma Cell Culture and Decitabine Treatment

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The cell culture adapted Eμ-Myc lymphoma cell clone 152M was maintained in RPMI 1640 medium (Sigma-Aldrich, Taufkirchen, Germany) supplemented with 10% (v/v) fetal calf serum (FCS), 50 µM β-mercaptoethanol (Carl Roth, Darmstadt, Germany), 10 mM HEPES and 100 units/mL penicillin and 0.1 mg/mL streptomycin (both Sigma). Cells were cultured under humidified atmosphere at 5% CO₂ and 37 °C. For inhibition of DNA methyltransferases, cells were treated with 0.1, 0.5, and 1.0 µM Decitabine (MedChemExpress, Monomouth Junction, NJ, USA) for 48 h. Cell viability was monitored by Trypan Blue (Sigma-Aldrich, Taufkirchen, Germany) staining.

Joshi G., Eberhardt A.O., Lange L., Winkler R., Hoffmann S., Kosan C, & Bierhoff H. (2020). Dichotomous Impact of Myc on rRNA Gene Activation and Silencing in B Cell Lymphomagenesis. Cancers, 12(10), 3009.

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NHBE Cell Culture and Stimulation

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NHBE culture was performed as previously described (40 (link)). Briefly, primary NHBEs (Lonza, Basel, Switzerland) of four genetically independent donors were grown as monolayers in 100% humidity and 5% CO₂ at 37 °C in serum-free defined growth media (BEGM, Lonza). NHBEs (passage 3) were used at ∼80% confluence in 6-well plates. To avoid gene expression changes or influences by growth factors in the BEGM medium, cells were rested in basal medium (BEBM, Lonza) for 12 h, then stimulated with HDM extract at a final concentration of 40µg/mL (CITEQ), recombinant human IL-4 at 50 ng/ml (R&D Systems, Minneapolis, MN, USA) for 6 h. When indicated, cells were pretreated for two h prior to stimulation with the AhR inhibitor CH-223191 (Sigma-Aldrich) at a concentration of 1µM/mL. For RNA analysis, harvested cells were lysed in RLT buffer (Qiagen, Hilden, Germany) containing 1% β-mercaptoethanol (Roth, Karlsruhe, Germany) directly in the cell culture well.

Alessandrini F., de Jong R., Wimmer M., Maier A.M., Fernandez I., Hils M., Buters J.T., Biedermann T., Zissler U.M., Hoffmann C., Esser-von-Bieren J., Schmidt-Weber C.B, & Ohnmacht C. (2022). Lung Epithelial CYP1 Activity Regulates Aryl Hydrocarbon Receptor Dependent Allergic Airway Inflammation. Frontiers in Immunology, 13, 901194.

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Culturing Liver Cell Lines

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Liver cell lines Huh7 and Sk-Hep-1 (kindly provided by P.
Knolle, Technische Universität München) were grown in DMEM (Sigma-Aldrich)
supplemented with 10% Fetal calf serum (FCS, Sigma-Aldrich) and 1%
penicillin-streptomycin (Lonza). Sk-Hep1 cells were additionally maintained
in 40 μM β-mercaptoethanol (Carl Roth). LX-2 cells were
provided by SL. Friedman (Icahn School of Medicine) and cultured in DMEM
high glucose (Life Technologies) supplemented with 2% FCS, 1%
penicillin-streptomycin. All cells were grown at 37 °C
under 5% CO₂.

Lee J.H., Ostalecki C., Zhao Z., Kesti T., Bruns H., Simon B., Harrer T., Saksela K, & Baur A.S. (2018). HIV Activates the Tyrosine Kinase Hck to Secrete ADAM Protease-Containing Extracellular Vesicles. EBioMedicine, 28, 151-161.

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Isolation and Stimulation of PBMCs

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Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation (density: 1.077 g/ml; PanBiotech) and washed twice with DPBS (Gibco). Cells were resuspended in FBS (Gibco) containing 10% DMSO (Merck) and gradually frozen to -180 °C. For cell culture experiments, PBMCs were thawed, washed twice with DPBS, resuspended in B cell medium consisting of IMDM with L-Glutamine and 25 mM HEPES supplemented with 10% FBS, 1% Penicillin/Streptomycin, 1 mM Sodium Pyruvate, 1% MEM non-essential aminoacids (all from Gibco) and 0.055 mM β-Mercaptoethanol (Carl Roth) and seeded at a concentration of 0.25 × 10⁶ cells/well into 96-well round bottom plates. Cells were rested for 2 h at 37 °C and 5% CO₂ in a humified incubation chamber, if not differently described.
To analyze the effect of D₁-like receptor stimulation on cytokine release, 0.25 × 10⁶ PBMCs were seeded per well of a 96-well round bottom plate and stimulated with CpG ODN 2006 (0.35 μM, InvivoGen) with or without indicated concentrations of the agonists A68930 (Tocris) and SKF38393 (Tocris) for 24 h. Afterwards, cells were centrifuged and supernatants were frozen at − 80 °C until analysis.

Wieber K., Fleige L., Tsiami S., Reinders J., Braun J., Baraliakos X, & Capellino S. (2022). Dopamine receptor 1 expressing B cells exert a proinflammatory role in female patients with rheumatoid arthritis. Scientific Reports, 12, 5985.

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Culturing Liver Cell Lines Huh7 and Sk-Hep1

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Liver cell lines Huh7 and Sk-Hep1 (kindly provided by P. Knolle, Technische Universität München) were grown in DMEM (Sigma-Aldrich) supplemented with 10% FCS (Sigma-Aldrich) and 1% penicillin–streptomycin (Lonza). Sk-Hep1 cells were additionally maintained in 40 μM β-mercaptoethanol (Carl Roth). LX-2 cells were provided by SL. Friedman (Icahn School of Medicine, New York) and cultured in DMEM high glucose (Life Technologies) supplemented with 2% FCS, 1% penicillin–streptomycin. All cells were grown at 37°C under 5% CO₂.

Lee J.H., Dindorf J., Eberhardt M., Lai X., Ostalecki C., Koliha N., Gross S., Blume K., Bruns H., Wild S., Schuler G., Vera J, & Baur A.S. (2019). Innate extracellular vesicles from melanoma patients suppress β-catenin in tumor cells by miRNA-34a. Life Science Alliance, 2(2), e201800205.

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HCMV-specific T Cell Isolation

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Buffy coats were kindly provided by the Institute for Clinical and Experimental Transfusion Medicine at the University Hospital of Tübingen after obtaining written informed consent in accordance with the Declaration of Helsinki and applicable laws and regulations. This has been approved by the Ethik-Kommission an der Medizinischen Fakultät der Eberhard-Karls-Universität und am Universitätsklinikum Tübingen (Project No. 507/2017BO1). Two-digit HLA-A and -B typing and CMV status were provided by Transfusion Medicine. Thereafter, blood samples were encoded by four-digit numbers. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy HCMV-seropositive blood donors by Ficoll-Hypaque density gradient centrifugation. Cells were frozen at −80°C in FCS and 10% DMSO. After thawing, cells were rested overnight before stimulation. Culture conditions were 7.5% CO₂ and 37°C in humidified incubators in IMDM (Thermo Fisher Scientific; 12440) supplemented with 5% heat-inactivated pooled human plasma (isolated from healthy blood donors), 100 U/ml penicillin, 100 µg/ml streptomycin, 25 µg/ml gentamicin (Life Technologies), and 50 µM β-mercaptoethanol (Carl Roth).

Lübke M., Spalt S., Kowalewski D.J., Zimmermann C., Bauersfeld L., Nelde A., Bichmann L., Marcu A., Peper J.K., Kohlbacher O., Walz J.S., Le-Trilling V.T., Hengel H., Rammensee H.G., Stevanović S, & Halenius A. (2019). Identification of HCMV-derived T cell epitopes in seropositive individuals through viral deletion models. The Journal of Experimental Medicine, 217(3), jem.20191164.

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Murine Blood and Immune Cell Isolation

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After mice were sacrificed by CO₂ inhalation, whole venous blood was collected and allowed to clot at room temperature. The clot was removed by centrifugation and serum was subsequently stored at −20 °C. Spleen and lymph nodes were retrieved, disintegrated mechanically and filtered through a 70-μm nylon Falcon^® cell strainer (Corning Life Sciences, Amsterdam, The Netherlands). After washing the cells with PBS (PromoCell, Heidelberg, Germany) lysis of erythrocytes was performed for SPCs by suspending the pelleted SPCs in 0.2 % NaCl for 30 s followed by two wash steps with culture medium, consisting of RPMI 1640 (PromoCell) supplemented with 10 % FCS (Lonza, Cologne, Germany), 100 U/mL Penicillin/100 μg/mL Streptomycin (PAA, Coelbe, Germany), 1 % l-glutamine (PAA) and 50 μM β-mercaptoethanol (Carl Roth, Karlsruhe, Germany).

Kleist C., Mohr E., Gaikwad S., Dittmar L., Kuerten S., Platten M., Mier W., Schmitt M., Opelz G, & Terness P. (2016). Autoantigen-specific immunosuppression with tolerogenic peripheral blood cells prevents relapses in a mouse model of relapsing-remitting multiple sclerosis. Journal of Translational Medicine, 14, 99.

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Modulation of AMPK Signaling Pathways

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M199 and Eagle’s minimum essential medium (EMEM) were purchased from Lonza (Basel, Switzerland). Fetal calf serum (FCS), human serum, endothelial cell supplement (ECGS), trypsin-EDTA, non-essential amino acids, L-glutamine, anhydrous dimethylsulfoxide (DMSO), Akt inhibitor VIII (Akt VIII), Torin-2, and Hoechst 33342 were from Sigma (Taufkirchen, Germany). bovine serum albumin-C (BSA-C) was obtained from Aurion (Wageningen, The Netherlands) and goat serum from Cell Signaling Technology (Frankfurt, Germany). EDTA-free protease inhibitor cocktail was purchased from Roche Diagnostics (Mannheim, Germany). β-Mercaptoethanol, dithiothreitol (DTT), EDTA, bovine serum albumin, Tween-20, and Triton X-100 were obtained from Carl Roth GmbH (Karlsruhe, Germany). Fluoromount-G was from Southern Biotech (Birmingham, AL, US). MK-8722 was purchased from Aobious (Gloucester, MA, USA). The siRNAs against AMPKα1 or AMPKα2, ACC1, and non-targeting control-siRNA were SMARTpool-siRNAs obtained from Horizon, Dharmacon RNAi, and Gene Expression (Lafayette, CO, USA).

Doshi H., Spengler K., Godbole A., Gee Y.S., Baell J., Oakhill J.S., Henke A, & Heller R. (2023). AMPK protects endothelial cells against HSV-1 replication via inhibition of mTORC1 and ACC1. Microbiology Spectrum, 11(5), e00417-23.

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