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β mercaptoethanol β me

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β-Mercaptoethanol (β-ME) is a reducing agent commonly used in molecular biology and biochemistry applications. It functions by breaking disulfide bonds in proteins, which can help denature and solubilize proteins for further analysis or processing.

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Lab products found in correlation

Rpmi 1640, by Thermo Fisher Scientific (3 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions) L glutamine, by Thermo Fisher Scientific (3 mentions) Hepes, by Thermo Fisher Scientific (3 mentions) Ni nta resin, by Thermo Fisher Scientific (3 mentions) E coli xl 10 cells, by Agilent Technologies (3 mentions) Deoxynucleotide mix pcr grade, by Agilent Technologies (3 mentions) Acetonitrile hplc grade, by Agilent Technologies (3 mentions) Pfuultra hotstart dna polymerase, by Agilent Technologies (3 mentions) Bovine serum albumin (bsa), by Merck Group (3 mentions) Lb broth, by Merck Group (3 mentions) Quikchange 2 site directed mutagenesis kit, by Agilent Technologies (3 mentions) Penicillin streptomycin glutamine (psg), by Thermo Fisher Scientific (2 mentions) Trizol, by Thermo Fisher Scientific (2 mentions) Sodium pyruvate, by Thermo Fisher Scientific (2 mentions) Thiamine pyrophosphate, by Merck Group (2 mentions) 1 deoxy d xylulose 5 phosphate sodium salt, by Merck Group (2 mentions) Poly l lysine (pll), by Merck Group (1 mentions) Lipopolysaccharide, by Merck Group (1 mentions) 96 well flat bottomed plate, by Corning (1 mentions)

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β-mercaptoethanol (1654 citations) ß-mercaptoethanol (100 citations)

12 protocols using β mercaptoethanol β me

1

Murine Bone Marrow-Derived Dendritic Cells

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Primary DCs were generated by isolating bone marrow cells from femurs and tibias of mice, followed by the elimination of erythrocytes. Bone marrow cells were cultured in very low endotoxin-DMEM (Biochrom) supplemented with 10% (v v−1) lipopolysaccharide (LPS)-free FCS (Biochrom); 1% (v v−1) penicillin-streptomycin-glutamine (Thermo Fisher Scientific); 0.1% (v v−1) 50 mM β-mercaptoethanol (β-ME) (Invitrogen); and 10 ng ml−1 granulocyte–macrophage colony-stimulating factor (made in-house). Cells were treated with lipopolysaccharide (100 ng ml−1, Sigma-Aldrich) for 24 h on day 6. On day 7 after harvesting, cells were washed with Dulbecco’s phosphate-buffered saline (DPBS), and 5 × 105 cells were transferred into a 24-well plate containing coverslips coated with poly-l-lysine (Sigma-Aldrich).
Khairnar V., Duhan V., Patil A.M., Zhou F., Bhat H., Thoens C., Sharma P., Adomati T., Friendrich S.K., Bezgovsek J., Dreesen J.D., Wennemuth G., Westendorf A.M., Zelinskyy G., Dittmer U., Hardt C., Timm J., Göthert J.R., Lang P.A., Singer B.B, & Lang K.S. (2018). CEACAM1 promotes CD8+ T cell responses and improves control of a chronic viral infection. Nature Communications, 9, 2561.
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2

Thymocyte Cultivation and Dexamethasone Treatment

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Female wild type (WT) or lckpr-bcl-xLtransgenic (TG) mice of 4–8 weeks of age were sacrificed by cervical dislocation, thymi were excided, thymocytes were reduced to single cell suspencion and counted with a hemocytometer. Single cell suspensions were cultured at a concentration of 3 ​× ​106 in Roswell Park Memorial Institute (RPMI) 1640 medium containing 5% fetal calf serum (FCS), 100 U/mL penicillin/streptomycin, 0.1 ​mM non-essential amino acids, 5.5 ​× ​102 ​μM β-mercaptoethanol (β-ME; GIBCO Invitrogen, San Giuliano Milanese, Italy) in flat-bottomed, 96-well plates (Costar, Cambridge, MA, USA) with or without 10−7 ​M dexamethasone (DEX).
Muscari I., Adorisio S., Liberati A.M., Thuy T.T., Van Sung T., Cannarile L., Ayroldi E., Riccardi C, & Delfino D.V. (2020). Bcl-xL overexpression decreases GILZ levels and inhibits glucocorticoid-induced activation of caspase-8 and caspase-3 in mouse thymocytes. Journal of Translational Autoimmunity, 3, 100035.
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3

Generating Primary Macrophages for LCMV Study

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Primary macrophages were generated by isolating bone marrow from femurs and tibias of mice and eliminating erythrocytes. Macrophages were generated by culturing bone marrow cells in very low endotoxin Dulbecco's modified Eagle's medium (VLE-DMEM) (Biochrom, Berlin, Germany) supplemented with 10% (v/v) fetal bovine serum (Biochrom), 0.1% (v/v) β-mercaptoethanol (β-ME) (Invitrogen, Carlsbad, CA, USA) and 10 ng/ml macrophage colony-stimulating factor (made in house). Cells were treated with 100 units of IFN-α4 (PBL Assay Science, Pistcataway, NJ, USA) on day 7 for 12 h. On day 8 of harvesting, cells were washed and 8 × 106 cells were transferred into LCMV-infected mice. Cells transfer was performed twice on day 5 and day 7 of infection.
Shaabani N., Duhan V., Khairnar V., Gassa A., Ferrer-Tur R., Häussinger D., Recher M., Zelinskyy G., Liu J., Dittmer U., Trilling M., Scheu S., Hardt C., Lang P.A., Honke N, & Lang K.S. (2016). CD169+ macrophages regulate PD-L1 expression via type I interferon and thereby prevent severe immunopathology after LCMV infection. Cell Death & Disease, 7(11), e2446-.
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4

Murine Dendritic Cell Isolation and Transcriptional Analysis

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Bone marrow was harvested from murine femurs and tibias. Erythrocytes were lysed with ACK lysis buffer, and dendritic cells were generated by culturing bone marrow cells in VLE-DMEM (Sigma, MO, USA) including 10% (v/v) fetal bovine serum (FBS) (Thermo Fisher; MA, USA), 0.1% (v/v) β-mercaptoethanol (β-ME) (Invitrogen, CA, USA), and 10 ng/ml granulocytes macrophage colony-stimulating factor (GM-CSF) (made in-house).
Total RNA extraction, c-DNA synthesis, and quantitative real-time PCR RNA was isolated from splenocytes or sorted cells with TRIzol (Thermo Fisher) as described in the manufacture's protocol. The RNA was reverse-transcribed into cDNA with the Quantitect Reverse Transcription Kit (Qiagen, Germany). Gene expression analysis was performed with assays from Qiagen: glyceraldehyde 3-phosphate dehydrogenase (GAPDH; QT01658692), Ifn-α4 (QT01774353), Ifn-β1 (QT00249662), Stat1 (QT00162183), Stat2 (QT00160216), Irf7 (QT00245266), Prkr (QT00162715), Usp18 (QT00167671), Ifnar2 (QT00102340), Oas1 (QT01056048), and Isg15 (QT02274335). Relative quantities (RQs) were determined with the equation RQ = 2 -ddCt .
Shaabani N., Vartabedian V.F., Nguyen N., Honke N., Huang Z, & Teijaro J.R. (2021). IFN-β, but not IFN-α, is Responsible for the Pro-Bacterial Effect of Type I Interferon. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, 55(3).
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5

KRAS Peptide Immunization in Transgenic Mice

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HLA-A*11:01 transgenic mice were injected subcutaneously at the base of the tail and footpads with KRAS G12V7-16 or KRAS G12D7-16, and helper peptide HBVc128-140 emulsified in incomplete Freund’s adjuvant (Sigma). Mice were immunized twice with KRAS G12V7-16, or three times with KRAS G12D7-16, with at least a 2-week interval between immunizations. Seven days after the final immunization, splenic and lymph node lymphocytes were harvested, pulsed with corresponding peptides at concentrations of 1 μM, 0.1 μM, or 0.01 μM, and then cultured in a 24-well plate at concentration of 3 × 106/ml in 2 ml of mouse T-cell medium including RPMI1640 plus 10% FBS, non-essential amino acid (Life Technologies), serum pyruvate (Life Technologies), β-mercaptoethanol (β-ME; Life Technologies) and recombinant human interleukin 2 (rhIL2; 30 IU/ml). Cell growth was monitored daily, and cultures split or replenish with fresh mouse T cell medium and rhIL2 when necessary. Seven days after in vitro stimulation, effector T cells (1 × 105) were cocultured with appropriate target cells (5 × 104) overnight, and the supernatant was harvested for IFNγ measurement by ELISA.
Wang Q.J., Yu Z., Griffith K., Hanada K.I., Restifo N.P, & Yang J.C. (2015). Identification of T-cell Receptors Targeting KRAS-mutated Human Tumors. Cancer immunology research, 4(3), 204-214.
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6

Murine Intestinal Organoid Culture

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Neutralizing antibody against Ly6G (1A8) was purchased from Bio × Cell. Recombinant AREG was purchased from Leinco Tecnologies. Thioglycollate broth and percoll were purchased from Sigma-Aldrich. Culture medium RPMI 1640, HEPES, penicillin/streptomycin, β-Mercaptoethanol (β-ME), sodium pyruvate, L-glutamine and ITS were purchased from Life Technologies. HBSS and DMEM were purchased from Corning. DMEM/F12 media (12634–010), L-glutamine (25030) and B27 (12587–010) were purchased from Invitrogen. Mouse recombinant cytokines were purchased from Biolegend. Retinoic acid was purchased from Sigma-Aldrich. Matrigel was purchased from BD Bioscience. Recombinant EGF (2028-EG), Noggin (1967-NG/CF), R-spondin (3474-RS), Wnt3a (35036-WN/CF) and N2 supplement (AR009) were purchased from R&D Systems. SMARTpool siRNAs specific for murine MEK1, MEK2 and non-targeting siRNA were purchased from Dharmacon. Inhibitor U0126 and PD98059 were purchased from Promega. Western blot antibodies against phosphorylated ERK1/2, β-actin, and anti-rabbit secondary antibody conjugated with HRP were purchased from Cell Signaling Technology.
Chen F., Yang W., Huang X., Cao A.T., Bilotta A.J., Xiao Y., Sun M., Chen L., Ma C., Liu X., Liu C.G., Yao S., Dann S.M., Liu Z, & Cong Y. (2018). Neutrophils promote amphiregulin production in intestinal epithelial cells through TGFβ and contribute to intestinal homeostasis. Journal of immunology (Baltimore, Md. : 1950), 201(8), 2492-2501.
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7

Th2 Cell Differentiation Protocol

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RPMI 1640, HEPES, penicillin/streptomycin, β-Mercaptoethanol (β-ME), sodium pyruvate and L-glutamine were all purchased from Life Technologies. Anti-TGF-β and control IgG were from Bioxcell. Th2-polarizing cytokines were from R&D Systems. Anti-mCD3 and fluorochrome-conjugated anti-mouse antibodies were all from Biolegend.
Wu W., Liu H.P., Chen F., Liu H., Cao A.T., Yao S., Sun M., Evans-Marin H.L., Zhao Y., Zhao Q., Duck L.W., Elson C.O., Liu Z, & Cong Y. (2016). Commensal A4 bacteria inhibit intestinal Th2-cell responses through induction of dendritic cell TGF-β production. European journal of immunology, 46(5), 1162-1167.
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8

Culturing Murine Embryonic Stem Cells

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Murine embryonic stem cells (mESCs; CGR8 strain) were obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK, No. 95011018). Cells were maintained in the medium for CGR8 (mCM) consisting of Glasgow’s minimum essential medium (Life Technologies, Darmstadt, Germany, No 11710035) with 2 mmol/L glutamine, 50 μmol/L β-mercaptoethanol (β-ME; Life Technologies, Darmstadt, Germany), 1000 units/mL leukemia inhibitory factor (LIF; Merck Chemicals, Darmstadt, Germany), penicillin (100 units/mL)/streptomycin (100 μg/mL) and 10% fetal bovine serum (FBS; GIBCO, Life Technologies, Darmstadt, Germany) at standard conditions (37 °C, 95% air and 5% CO2 atmosphere). Cell culture flasks (25 cm2, Nunc, Novodirect, Kehl, Germany) were coated with 2 mL of 0.2% gelatin and incubated for 30 min (5% CO2, 37 °C). After incubation, the gelatin was aspirated and 5 mL of mCM containing 1 × 106 mESCs was added to each flask. Cells were passaged at the sub-confluent stage (70–80%). The medium was exchanged after 2–3 days.
Hellweg C.E., Shinde V., Srinivasan S.P., Henry M., Rotshteyn T., Baumstark-Khan C., Schmitz C., Feles S., Spitta L.F., Hemmersbach R., Hescheler J, & Sachinidis A. (2020). Radiation Response of Murine Embryonic Stem Cells. Cells, 9(7), 1650.
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9

Molecular Mechanisms of miR-107 Regulation

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RPMI 1640, HEPES, penicillin/streptomycin, β-Mercaptoethanol (β-ME), sodium pyruvate and L-glutamine were purchased from Life Technologies (Carlsbad, CA). GM-CSF, anti-CD11c, anti-CD4, and anti-B220 were from BD Biosciences (San Diego, CA). Restriction endonucleases and T4 DNA ligase were from New England Biolabs (Ipswich, MA). The psiCHECK-2 vector and the dual-luciferase reporter system were from Promega (Madison, WI). Lipofectamine 2000 was purchased from Invitrogen (Carlsbad, CA). Collagenase IV was obtained from Sigma-Aldrich (St. Louis, MO). MiR-107 precursors and inhibitors were purchased from Ambion (Carlsbad, CA) and TaqMan® microRNA reverse transcription kit and gene expression assays from Applied Biosystems (Carlsbad, CA). Nucleotides were synthesized by Fisher Scientific (Pittsburgh, PA).
Xue X., Cao A.T., Cao X., Yao S., Carlson E.D., Soong L., Liu C.G., Liu X., Liu Z., Duck L.W., Elson C.O, & Cong Y. (2014). Downregulation of MicroRNA-107 in intestinal CD11c+ myeloid cells in response to microbiota and proinflammatory cytokines increases IL-23p19 expression. European journal of immunology, 44(3), 673-682.
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10

Recombinant Enzyme Production Protocol

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Thiamine pyrophosphate (TPP), pyruvate, D,L-glyceraldehyde-3-phosphate (GAP), 1-deoxy-D-xylulose-5-phosphate sodium salt, bovine serum albumin, and LB-broth were purchased from Sigma Aldrich. NADPH was purchased from Alexis Biochemical, Ni-NTA resin was purchased from Invitrogen, and β-mercaptoethanol (β-Me) was purchased from Fisher. E. coli XL-10 cells, deoxynucleotide mix PCR grade, pfuUltra Hotstart DNA polymerase, QuikChange II site directed mutagenesis kit and acetonitrile (HPLC grade) were purchased from Agilent. The DNA vectors, pET28a (+) and pET15b (+), and E. coli BL-21 B (DE3) cells were purchased from EMD Biosciences. DNA sequencing services and primers were purchased from MWG operon. All the other reagents were of the highest quality commercially available.
Handa S., Dempsey D.R., Ramamoorthy D., Cook N., Guida W.C., Spradling T.J., White J.K., Woodcock H.L, & Merkler D.J. (2018). Mechanistic Studies of 1-Deoxy-D-Xylulose-5-Phosphate Synthase from Deinococcus radiodurans. Biochemistry & molecular biology journal, 4(1), 2.
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