β mercapthoethanol

Manufactured by Merck Group

Sourced in Germany

β-mercapthoethanol is a colorless, viscous liquid used in various laboratory applications. It functions as a reducing agent, inhibiting oxidation and preserving the structural integrity of biomolecules.

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10 protocols using β mercapthoethanol

Isolation and Activation of Peritoneal B1a Cells

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Peritoneal B1a cells were obtained through negative magnetic-activated cell separation with a cocktail of antibodies depleting other than B1a cells achieving more than 90% purity in isolation (Miltenyi Biotec B.V., Utrecht, The Netherlands). B1a cells were subsequently counted and plated in IMDM medium (Invitrogen, Eugene, OR, USA) supplemented with 10% fetal calf-serum (FCS; Bodinco, Alkmaar, The Netherlands, 100 U/ml of penicillin, 100 mg/ml of streptomycin, and 2 mM L-glutamine (all Gibco Invitrogen, Breda, The Netherlands), and beta-mercapthoethanol (3.57 × 10^–4M; Millipore, Amsterdam, The Netherlands). Cells were plated in 96-well plate in density of 1 × 10⁶/ml in 200 μl of medium and cultured at 37°C and 5% CO2 for 48h in presence of 5 μg of lipopolysaccharide (LPS, isolated from E. coli strain 055:B5, Sigma, St. Louis, MO, USA; LBP from R&D Systems, Abingdon, UK), isotype control (rat IgG2b, eBioscience, San Diego, CA, USA), anti-CD11b antibody (functional grade, eBioscience, San Diego, CA, USA), or anti-CD11a (functional grade, eBioscience, San Diego, CA, USA) in final concentration 10 μg/ml. After that supernatant was collected and stored at −80°C before measurement of IgM antibody by ELISA. Cells were harvested and processed for analysis by flow cytometry and imaging cytometry.

Franke K., Pillai S.Y., Hoogenboezem M., Gijbels M.J., Matlung H.L., Geissler J., Olsman H., Pottgens C., van Gorp P.J., Ozsvar-Kozma M., Saito Y., Matozaki T., Kuijpers T.W., Hendriks R.W., Kraal G., Binder C.J., de Winther M.P, & van den Berg T.K. (2020). SIRPα on Mouse B1 Cells Restricts Lymphoid Tissue Migration and Natural Antibody Production. Frontiers in Immunology, 11, 570963.

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Isolation and Activation of Peritoneal B1a Cells

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Peritoneal B1a cells were obtained through negative magnetic-activated cell separation with a cocktail of antibodies depleting other than B1a cells achieving more than 90% purity in isolation (Miltenyi Biotec B.V., Utrecht, The Netherlands). B1a cells were subsequently counted and plated in IMDM medium (Invitrogen, Eugene, OR, USA) supplemented with 10% fetal calfserum (FCS; Bodinco, Alkmaar, The Netherlands, 100 U/mL of penicillin, 100 mg/mL of streptomycin, and 2 mM L-glutamine (all Gibco Invitrogen, Breda, The Netherlands), and betamercapthoethanol (3.57 x 10 -4 M; Millipore, Amsterdam, the Netherlands). Cells were plated in 96-well plate in density of 1x10 6 /mL in 200L of medium and cultured at 37C and 5% CO2 for 48h in presence of 5g of lipopolysaccharide (LPS, isolated from E. coli strain 055:B5, Sigma, St. Louis, MO, USA; LBP from R&D Systems, Abingdon, UK), isotype control (rat IgG2b, eBioscience, San Diego, CA, USA), anti-CD11b antibody (functional grade, eBioscience, San Diego, CA, USA), or anti-CD11a (functional grade, eBioscience, San Diego, CA, USA) in final concentration 10g/mL. After that supernatant was collected and stored at -80C before measurement of IgM antibody by ELISA. Cells were harvested and processed for analysis by flow cytometry and imaging cytometry.

Franke K., Pillai S.Y., Hoogenboezem M., Gijbels M.J., Matlung H.L., Geissler J., Olsman H., Pöttgens C., Gorp P.J.v., Ozsvár-Kozma M., Saito Y., Matozaki T., Kuijpers T.W., Hendriks R.W., Kraal G., Binder C.J., Winther M.P.d., & Berg T.K.v.d. (2020). SIRPα on mouse B1 cells restricts lymphoid tissue migration and natural antibody production.

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Tubulin Labeling and Modification Protocol

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Tubulin was purified from bovine brain tissue according to the established protocol (Castoldi and Popov, 2003 (link)). We conjugated two fluorescent dyes: Alexa Flour 568 NHS Ester (Life Technologies, A-20006), Alexa Flour 647 NHS Ester (Life Technologies, A-20003) orbiotin-PEG-NHS (Thermo Scienti1c, 20217) to tubulin as described previously (Hyman et al., 1991 (link)). We prepared NEM-modified tubulin by incubation of unmodified dimers at a concentration of 13 mg/ml with 1 mM NEM (N-Ethylmaleimide, Sigma, E3876) and 0.5 mM GMPCPP (Jena Bioscience, NU-405S) on ice for 10 min and then quenching the reaction with 8 mM beta-mercapthoethanol (Sigma, M6250) for another 10 mins (Hyman et al., 1991 (link)). All labeled and unlabeled tubulin monomers were stored at −80

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Memet E., Hilitski F., Morris M.A., Schwenger W.J., Dogic Z, & Mahadevan L. (2018). Microtubules soften due to cross-sectional flattening. eLife, 7, e34695.

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Genetic Manipulation of Naïve B Cells

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Naïve B cells from Rosa26^LSL-Cas9 and C57BL/6 mice were isolated by CD43 depletion using CD43 microbeads (Miltenyi Biotec). B cells were cultured at 1x10⁶ cells/ml in DMEM medium supplied with 15 % FBS, 2 mM HEPES (Gibco), 2 mM Sodium Pyruvate (Gibco), 2 mM L-Glutamine (Gibco), and 1x NAA (Gibco), beta-mercapthoethanol (Sigma) and stimulated with LPS (10 μg/ml). In addition, 5–10x10⁶ naïve B cells isolated from Rosa26^LSL-Cas9 mice were treated with TAT-Cre protein as previously described [31 (link)]. Briefly, CD43-depleted B cells were washed 3 times with HyClone™ ADCF-Mab medium (GE Heathcare), incubated with TAT-Cre for 45 min at 37 °C, finally the cells were washed with complete medium. TAT-Cre-treated B cells were stimulated with LPS for 2 or 3 days.

Chu V.T., Weber T., Graf R., Sommermann T., Petsch K., Sack U., Volchkov P., Rajewsky K, & Kühn R. (2016). Efficient generation of Rosa26 knock-in mice using CRISPR/Cas9 in C57BL/6 zygotes. BMC Biotechnology, 16, 4.

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Western Blot Analysis of Mutant Embryos

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16–18 ss mutant embryos (visually identified) and stage-matched WT embryos were dechorionated and collected in separate Eppendorf tubes. A total of 20 embryos from each group were pooled into one tube to constitute a biological replicate. In 1× PBS, embryos were mechanically de-yolked with a pipette tip. Samples were washed in cold 1× PBS thrice, and then 4× Laemmli sample buffer (Bio-Rad) with B-mercapthoethanol (Sigma) was added. Samples were vortexed and boiled at 95°C for 5 min three times. Samples were stored in –20°C until ready for use.
Proteins from each sample were resolved using a precast 8–16% gradient gel (Bio-Rad), and transferred to a PVDF membrane following the manufacturer’s protocol. Membranes were blocked with 3–5% BSA in 1× TBST (1× TBS, 0.5% Tween-20) for an hour, followed by incubation with primary antibody overnight at 4°C. Membranes were washed with 1× TBST before incubation with secondary antibody for at least 1 hr. Membranes were washed again with 1× TBST. ECL substrate (Bio-Rad) was added to the membrane and imaged with the ChemiDoc imaging system (Bio-Rad).
The primary antibodies used in this assay are: anti-B-actin (A5441, Sigma), 1:1000; anti-γH2AX (GTX127342, Genetex), 1:1000.
The secondary antibodies used in this assay are: HRP-conjugated anti-mouse (31430, Thermo Fisher), 1:10,000; HRP-conjugated anti-rabbit (31460, Thermo Fisher), 1:10,000.

Lai J.K., Toh P.J., Cognart H.A., Chouhan G, & Saunders T.E. (2022). DNA-damage induced cell death in yap1;wwtr1 mutant epidermal basal cells. eLife, 11, e72302.

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Western Blot Analysis of Mutant Embryos

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16-18 ss mutant embryos (visually identified) and stage-matched WT embryos were dechorionated and collected in separate Eppendorf tubes. A total of 20 embryos from each group were pooled into one tube to constitute a biological replicate. In 1X PBS, embryos were mechanically de-yolked with a pipette tip. Samples were washed in cold 1X PBS thrice, and then 4X Laemmli Sample Buffer (Biorad) with B-mercapthoethanol (Sigma) was added. Samples were vortexed and boiled at 95°C for 5 minutes three times. Samples were stored in -20°C until ready for use.
Proteins from each sample were resolved using a precast 8-16% gradient gel (Biorad), and transferred to a PVDF membrane following manufacturer's protocol. Membranes were blocked with 3-5% BSA in 1X TBST (1X TBS, 0.5% Tween-20) for an hour, followed by incubation with primary antibody overnight at 4°C. Membranes were washed with 1X TBST before incubation with secondary antibody for at least 1 hour. Membranes were washed again with 1X TBST. ECL substrate (Biorad) was added to the membrane and imaged with the ChemiDoc imaging system (Biorad).
The primary antibodies used in this assay are: anti-B-actin (A5441, Sigma), 1:1000; anti-γH2AX (GTX127342, Genetex), 1:1000.
The secondary antibodies used in this assay are: HRP-conjugated anti-mouse (31430, Thermo Fisher), 1:10000; HRP-conjugated anti-rabbit (31460, Thermo Fisher), 1:10000.

Lai J., Toh P.J.Y., Cognart H.A., Chouhan G., & Saunders T.E. (2021). DNA-damage induced cell death in yap1;wwtr1 mutant epidermal basal cells.

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Isolation and Cultivation of Echinococcus multilocularis Metacestodes

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Echinococcus multilocularis metacestodes were isolated, separated from host contaminants and axenically cultivated as previously described (6 (link)). For the collection of E/S products, metacestode vesicles were kept under axenic conditions for 10 days, washed three times in 1 x PBS and resuspended in DMEM10redox i.e., DMEM ⁺ GlutamaxTM, GIBCO supplemented with 10% Fetal Bovine Serum Superior (Biochrom AG), 100 μg/ml penicillin/streptomycin (PenStrep solution, Biochrom AG), 20 μg/ml Levofloxacin (Tavanic, Sanofi-Aventis), β-mercapthoethanol (143 μM, Sigma-Aldrich, cat. M6250), 10 μM Bathocuproine disulfonic acid (Sigma, cat. B-1125) and 100 μM L-Cysteine (Sigma, cat. C-1276) under axenic conditions [see (6 (link)) for description of conditions]. After 48 h of culture, the supernatants containing the MVE/S were collected and filtered through a 0.2 μm sieve (Filtropur S filter, SARSTEDT). The total amount of E/S product proteins was determined as previously defined (6 (link)) and the E/S products stored at −80°C until use.

Nono J.K., Lutz M.B, & Brehm K. (2020). Expansion of Host Regulatory T Cells by Secreted Products of the Tapeworm Echinococcus multilocularis. Frontiers in Immunology, 11, 798.

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Mouse Embryonic Stem Cell Culture

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Mouse embryonic stem cells (ESCs) were cultured in EmbryoMax DMEM (EMD Millipore) supplemented with 15% embryonic stem cell screened fetal bovine serum (HyClone), 2mM L-glutamine (Life Technologies), 1x non-essential amino acids EmbryoMax MEM (EMD Millipore), 1x EmbryoMax nucleosides (EMD Millipore), 0.1mM β-mercapthoethanol (Sigma-Aldrich), 100U/ml penicillin-streptomycin (Life Technologies), 1000U/ml ESGRO Leukemia inhibitory factor (EMD Millipore), 2.5μM GSK-3 inhibitor XVI (EMD Millipore) and 50nM FGF receptor antagonist PD173074 (Tocris). NIH3T3 cells were grown in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific), 2mM glutamine (Life Technologies), and 100U/ml penicillin-streptomycin (Life Technologies). All cells were determined to be negative for mycoplasma using the Venor GeM Mycoplasma detection kit (Sigma-Aldrich).

Jacko M., Weyn-Vanhentenryck S.M., Smerdon J.W., Yan R., Feng H., Williams D.J., Pai J., Xu K., Wichterle H, & Zhang C. (2018). Rbfox splicing factors promote neuronal maturation and axon initial segment assembly. Neuron, 97(4), 853-868.e6.

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Isolation and Culture of Murine Dendritic Cells and T Cell Hybridomas

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Bone marrow-derived DCs were prepared from femurs/tibiae of mice between 6-12 weeks of age and cultured for 5-7 days with 2-3 medium replenishments without disturbing the cells. DCs were kept in RPMI 1640 (Sigma) with 10% FBS (Thermo, Fisher Scientific), 50 μM β-mercapthoethanol (Sigma) and 2mM L-glutamine, 100U/ml penicillin, 100mg/ml streptomycin (Pen/Strep), 12mM HEPES, non-essential amino acids (all GIBCO) and 20 ng/ml GM-CSF. Bone marrow-derived macrophages were prepared in the same fashion but incubated with RPMI-1640 containing M-CSF derived from L292 cells.
The B3Z CD8⁺ T cell hybridoma specific for OVA_257-264-associated H2-K^b and the BO4 hybridoma specific for HEL_74-88-associated I-A^b were grown in RPMI 1640 medium with 10% FBS, 2mM L-glutamine, Pen/Strep, 50μM 2-ME, 1mM pyruvate.
MHC class I-restricted T cells specific for the immunodominant peptide from HSV glycoprotein B (gB), gB_498-505, were purified from spleens of gBT mice using mouse CD8a (Ly-2) MicroBeads from Miltenyi Biotec according to the manufacturer's protocol. Two rounds of isolation were performed and more than 80% of cells isolated were CD3 (T cell surface marker) positive as shown by FACS analysis.
HeLa, HEK 293T, and Vero cells were grown in Iscove's Modified Dulbecco's Medium (IMDM) with 10% FBS, GlutaMax (GIBCO) and 100U/ml penicillin, 100mg/ml streptomycin (Pen/Strep).

Samie M, & Cresswell P. (2015). The transcription factor TFEB acts as a molecular switch that regulates exogenous antigen presentation pathways. Nature immunology, 16(7), 729-736.

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Total RNA Isolation and qRT-PCR Analysis

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For total RNA isolation, the RNeasy Plus mini kit (Qiagen, Germany) was used, according to manufacturer`s instructions. Tissue blocks were transferred to Lysing Matrix A tubes (MP Biomedicals, Germany) in RLT buffer supplemented with 1% β-Mercapthoethanol (Sigma, Germany). Tissue was lysed using FastPrep®-24 (MP Biomedicals, Germany) by applying six rounds of lysis at default settings (4 m/s, 20 s). Homogenates were centrifuged and RNA was purified from the supernatant. Total RNA was reverse transcribed with oligo (dT) primers and RevertAid H Minus Reverse Transcriptase (Thermo Fisher Scientific, USA). qRT-PCR was carried out in duplicates using a LightCycler 480 II (Roche, Germany) by using specific primer sequences (Supplementary Table 1). Commercial primers were used for analysis of IFITM3 and IFN-β mRNA (Qiagen, Germany). mRNA expression data were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and analysed by using the 2^−ΔΔCT method.

Matos A.D., Wunderlich K., Schloer S., Schughart K., Geffers R., Seders M., de Witt M., Christersson A., Wiewrodt R., Wiebe K., Barth P., Hocke A., Hippenstiel S., Hönzke K., Dittmer U., Sutter K., Rescher U., Rodionycheva S., Matera N., Ludwig S, & Brunotte L. (2019). Antiviral potential of human IFN-α subtypes against influenza A H3N2 infection in human lung explants reveals subtype-specific activities. Emerging Microbes & Infections, 8(1), 1763-1776.

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