M6250

Manufactured by Merck Group

Sourced in United States, Spain

The M6250 is a laboratory equipment product manufactured by Merck Group. It is a multi-purpose device designed for various applications in scientific and research settings. The core function of the M6250 is to perform precise measurements and analyses as required by the user's specific needs. No further details about its intended use or interpretation of its capabilities are provided.

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Lab products found in correlation

Rneasy mini kit, by Qiagen (4 mentions) P8783, by Merck Group (4 mentions) H 2270, by Merck Group (3 mentions) P 7505, by Merck Group (3 mentions) U3003, by Merck Group (3 mentions) L glutamine, by Thermo Fisher Scientific (3 mentions) Sodium bicarbonate, by Thermo Fisher Scientific (3 mentions) Hep001, by Bio Basic (2 mentions) Sb0476, by Bio Basic (2 mentions) Ndb0385, by Bio Basic (2 mentions) Sov664, by Bio Basic (2 mentions) Ad0153, by Bio Basic (2 mentions) Ldj691, by Bio Basic (2 mentions) Leupeptin, by Bio Basic (2 mentions) Mmv00, by R&D Systems (2 mentions) Gw9508, by Cayman Chemical (2 mentions) A4036, by Thermo Fisher Scientific (2 mentions) C4654, by Thermo Fisher Scientific (2 mentions) T1895, by Thermo Fisher Scientific (2 mentions) G6264, by Thermo Fisher Scientific (2 mentions)

Close spelling variants of this product

β-mercaptoethanol (M6250) (2 citations)

46 protocols using m6250

Culturing Embryonic Stem and HEK293T Cells

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ESCs were cultured on 0.1% gelatin-coated plates at 37°C with 5% CO₂ and maintained in DMEM (high glucose, D6429; Sigma-Aldrich), containing 15% FBS (10270-106; Gibco), 2 mM L-glutamine (G7513; Sigma-Aldrich), 1× nonessential amino acids (11140; Gibco), 1% nucleoside mix, 1× penicillin/streptomycin (15140; Gibco), 10⁻⁴M 2-mercaptoethanol (M6250; Sigma-Aldrich), and 1,000 U/ml of recombinant LIF.
HEK293T cells were cultured at 37°C with 5% CO₂ in DMEM (high glucose, D6429; Sigma-Aldrich), containing 10% FBS (10270-106; Gibco), 2 mM L-glutamine (SH30034.01; GE Healthcare), 1× penicillin/streptomycin (15140; Gibco), and 10⁻⁴ M 2-mercaptoethanol (M6250; Sigma-Aldrich).

Ameneiro C., Moreira T., Fuentes-Iglesias A., Coego A., Garcia-Outeiral V., Escudero A., Torrecilla D., Mulero-Navarro S., Carvajal-Gonzalez J.M., Guallar D, & Fidalgo M. (2020). BMAL1 coordinates energy metabolism and differentiation of pluripotent stem cells. Life Science Alliance, 3(5), e201900534.

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Dissociation and FACS Sorting of EHTs

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At cast week 1 or week 3, EHTs were rinsed with 1x PBS and carefully removed from PDMS posts using forceps. Tissues were placed in 1 mL of papain-based dissociation solution [40 U/mL papain from C. papaya (Sigma, 76220-25G), 5.5 mM L-cysteine HCl monohydrate (Sigma, C7880-500MG), 1 mM EDTA (Fisher, 02-002-790), 0.5% beta-mercaptoethanol (Sigma, M6250), 1x PBS] (28). Tissues in dissociation solution were incubated for 10 min at 37C, 5% CO2 and gently triturated into single cells. Dissociation was halted with 5% FBS in RPMI, and cells were centrifuged for 3 min at 300g and resuspended in 5% FBS in 1x PBS. 10% of cells were fixed with 4% paraformaldehyde for 5 min for replicate analysis by flow cytometry. The remaining cells were filtered with 40 µm filters, centrifuged for 3 min at 300g, and resuspended in 5% FBS in 1x PBS. Cells were sorted using a BD FACSAria II sorter with 70 μm nozzle, serially gating for FSC-A/SSC-A, FSC-H/FSC-W, and GFP+/AmCyan- populations. Sorted cells were collected in 5% FBS in 1x PBS for analysis.

Loiben A.M., Chien W.M., Friedman C.E., Chao L.S., Weber G., Goldstein A., Sniadecki N., Murry C.E, & Yang K.C. (2023). Cardiomyocyte apoptosis contributes to contractile dysfunction in stem cell model of MYH7 E848G hypertrophic cardiomyopathy. bioRxiv.

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Bladder Macrophage Transcriptome Analysis

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Sorted bladder macrophages were transferred to buffer RLT with β‐mercaptoethanol (Sigma, M6250), with at least 6 × 10³ cells being per sorted bladder cell population. RNA was then isolated from the cells and frozen, per the manufacturer's protocols (RNeasy Mini kit, Qiagen), and assessed for quality using the Bioanalyzer 2100 (Agilent). With the Sigma WTA2 RNA amplification kit, 1.0 ng of total RNA were amplified and then 3 µg of cDNA were chemically labeled with Kreatech ULS Fluorescent Labeling Kit for Agilent arrays (Kreatech Diagnostics). Finally, 1.7 µg of labeled cDNAs were subsequently hybridized onto Agilent Mouse Gene Expression 4 × 44 K Microarrays (cat. G4122F‐014868). Slides were scanned on an Agilent C‐class Microarray scanner to detect Cy5 fluorescence, according to manufacturer's specifications. Gridding and analysis of images was performed using Feature Extraction (v11.5.1.1, Agilent Technologies). The data was then analyzed, and normalized heat maps were generated, using the R software package, with a cutoff of P ≤ 0.05 and fold change of >2.

Owusu‐Boaitey N., Bauckman K.A., Zhang T, & Mysorekar I.U. (2016). Macrophagic control of the response to uropathogenic E. coli infection by regulation of iron retention in an IL‐6‐dependent manner. Immunity, Inflammation and Disease, 4(4), 413-426.

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Co-immunoprecipitation Workflow for Protein Interactions

Cited 1 time

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Co-immunoprecipitations were conducted according to the protocol in [77 (link)]. Briefly, the protein concentration of E⁻Du145 and E⁻PC3 lysates were determined by Lowry assay (Biorad Dc assay cat: 500, Biorad, UK). Aliquots of cell lysates containing 500 μg protein were transferred to new tubes and the volume adjusted to 400 μL with lysis buffer. 30 μL packed Pierce Protein A/G Plus Agarose Beads (Life Technologies #20423) were combined with 500 μg whole cell lysate on a rocker at 4 °C for 1 h to clear the lysate. 500 μg cleared protein lysate and 5 μg/mL primary antibody were rocked overnight at 4 °C. Control samples had 5 μg isotype-matched control IgG added. 30 μL of fresh packed beads were added to the protein lysate with primary antibody and rocked at 4 °C for 1 h. Samples were centrifuged for 5 min at 10,000 × g. The beads were washed three times with 500 μL immunoprecipitation buffer (20 mM Tris, 100 mM NaCl, 1 mM EDTA, 1% Tween) before applying 25 μL 2× SDS sample buffer (Bio-Rad 161–0737) with 10% β-mercaptoethanol (Sigma-Aldrich M6250) and boiled at 100 °C for 5 min. The samples were centrifuged briefly at room temperature before loading with Prot/Elec tips (Bio-Rad 2239916) on a western blot.

Pollan S.G., Huang F., Sperger J.M., Lang J.M., Morrissey C., Cress A.E., Chu C.Y., Bhowmick N.A., You S., Freeman M.R., Spassov D.S., Moasser M.M., Carter W.G., Satapathy S.R., Shah K, & Knudsen B.S. (2018). Regulation of inside-out β1-integrin activation by CDCP1. Oncogene, 37(21), 2817-2836.

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Western Blot Analysis of Mitochondrial Proteins

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Laemmli buffer containing 4% SDS, 10% β-mercaptoethanol (M6250; Sigma-Aldrich), 20% glycerol (GI345; Melford), 0.004% blue bromophenol (A2331,0025; AppliChem), and 0.125 M Tris-HCl was added to protein lysates, followed by boiling at 95°C for 10 min. Samples were separated on SDS-PAGE and transferred to a nitrocellulose (Macherey-Nagel) or a polyvinylidene difluoride (Merck) membrane. The membranes were blocked with 5% milk (A0830; PanReac AppliChem) and probed with the following antibodies: anti–β-Actin (ab8227; Abcam), anti–β-Tubulin (ab15568; Abcam), anti-CBS (14787-1-AP; Proteintech), anti-CSE (12217-1-AP; Proteintech), anti-MPST (HPA001240; Atlas Antibodies), anti-MTCO1 (ab14705; Abcam), anti-Citrate Synthase (ab96600; Abcam), anti-SOD2 (13141; Cell Signaling), anti-TOM40L (ab236421; Abcam), anti-TIM50 (ab109436; Abcam), anti-HIF1α (sc-13515; Santa Cruz Biotechnology), anti-H3 (ab176842; Abcam), anti-V5 (Merck), anti-HA (H6908; Sigma-Aldrich), and anti-Biotin (HRP conjugate; 5571; Cell Signaling). Immunoblots were next incubated with a secondary antibody (anti-rabbit [AP132P; Merck] or anti-mouse [7076; Cell Signaling]) and visualized using the Western HRP substrate (Merck). ImageJ software was used to quantify the expression levels of the proteins.

Katsouda A., Valakos D., Dionellis V.S., Bibli S.I., Akoumianakis I., Karaliota S., Zuhra K., Fleming I., Nagahara N., Havaki S., Gorgoulis V.G., Thanos D., Antoniades C., Szabo C, & Papapetropoulos A. (2022). MPST sulfurtransferase maintains mitochondrial protein import and cellular bioenergetics to attenuate obesity. The Journal of Experimental Medicine, 219(7), e20211894.

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Protein Extraction from Flash-Frozen Tumor Tissue

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Flash frozen tumor tissue was disrupted by mortar and pestle in liquid nitrogen, and lysed in a modified Hepes lysis buffer (50mM Hepes pH 7.5 (Bioshop HEP001), 150mM NaCl (Biobasic SB0476), 10% Glycerol (GB0232), 0.5% NP-40 (Biobasic NDB0385), 1mM EDTA pH 8.0 (Biobasic EB0185), 10mM NaF (Bioshop SFL001), 1mM PMSF (Bioshop PMS123), 10mM β Glycerophosphate (Bioshop GYP001), 1mM Na₃VO₄ (Bioshop SOV664), 10μg/ml Aprotinin (Biobasic AD0153), 10μg/ml Leupeptin (Biobasic LDJ691). Lysates were quantified by Bradford assay, and reduced in SDS loading buffer with β-mercaptoethanol (Sigma M6250). Immunoblots are representative of 3 independent experiments.

Turpin J., Ling C., Crosby E.J., Hartmann Z.C., Simond A.M., Chodosh L.A., Rennhack J.P., Andrechek E.R., Ozcelik J., Hallett M., Mills G.B., Cardiff R.D., Gray J.W., Griffith O.L, & Muller W.J. (2016). The ErbB2ΔEx16 splice variant is a major oncogenic driver in breast cancer that promotes a pro-metastatic tumor microenvironment. Oncogene, 35(47), 6053-6064.

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Extraction and Analysis of Insoluble Tau Proteins

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Matrigel embedded differentiated cells were harvested in sarkosyl lysis buffer supplemented with protease and phosphatase inhibitor cocktails (Thermo Fisher Scientific). Lysed samples were homogenized by pipetting, incubated on ice for 30 min, sonicated for 10 min at 4 °C, and centrifuged at 300,000 × g for 30 min at 4 °C. The supernatants containing sarkosyl-soluble fractions were collected. To prepare sarkosyl-insoluble fractions for Western blotting, the pellets were carefully rinsed twice with 100 μl of ice-cold PBS and dissolved in 1× LDS sample buffer (NP0007, Invitrogen) with 2% β-mercaptoethanol (M6250, Sigma-Aldrich). The samples were boiled at 95 °C for 5 min followed by Western blot analysis to detect insoluble tau proteins.

Kwak S.S., Washicosky K.J., Brand E., von Maydell D., Aronson J., Kim S., Capen D.E., Cetinbas M., Sadreyev R., Ning S., Bylykbashi E., Xia W., Wagner S.L., Choi S.H., Tanzi R.E, & Kim D.Y. (2020). Amyloid-β42/40 ratio drives tau pathology in 3D human neural cell culture models of Alzheimer’s disease. Nature Communications, 11, 1377.

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Cone Photoreceptor Cell Culture and Hypoxia Signaling

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Cone photoreceptor cells^{47 (link),48 (link)} (661W; from Dr. Al-Ubaidi) were cultured as monolayers at 37 °C, 5% CO₂ in a humidified atmosphere in DMEM with FBS 10% supplemented with hydrocortisone (20 µg/500 mL, H-2270, Sigma), Progesterone (20 µg/500 mL, P-8783, Sigma), Putrescine (0.016 g/500 mL, P-7505, Sigma) and β-mercaptoethanol (20 µL/500 mL, M-6250, Sigma). Cells were not contaminated by mycoplasma. Equal number of 661W cells (0.3×10⁶) were plated in 6-well dishes and cultured to 80% confluence. Cells were washed twice with PBS, starved for 4 hours (above medium without FBS) then stimulated with GW9508 (14 µM, Cayman) or vehicle. Photoreceptors were then collected 8 hours post-treatment for Hif1a protein expression (see western blot); while their medium was collected at 12 hours for Vegfa quantification by ELISA (as per manual, MMV00, R&D Systems). Vegfa concentration was normalized for the number of cells per well, by doing a Bradford to measure the total cell protein content of each well.

Joyal J.S., Sun Y., Gantner M.L., Shao Z., Evans L.P., Saba N., Fredrick T., Burnim S., Kim J.S., Patel G., Juan A.M., Hurst C.G., Hatton C.J., Cui Z., Pierce K.A., Bherer P., Aguilar E., Powner M.B., Vevis K., Boisvert M., Fu Z., Levy E., Fruttiger M., Packard A., Rezende F.A., Maranda B., Sapieha P., Chen J., Friedlander M., Clish C.B, & Smith L.E. (2016). Retinal lipid and glucose metabolism dictates angiogenesis through lipid sensor Ffar1. Nature medicine, 22(4), 439-445.

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Culturing murine embryonic stem cells

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R1 mESCs, ZHBTc4 (mESC lines) (Niwa et al., 2000 (link)) and pre-iPS cells were cultured on gelatin-coated tissue culture plates in knockout DMEM (Thermo Fisher, 10829018) supplemented with 15% fetal calf serum (Lonsera, S711-004S/NN02953), 1× nonessential amino acids (Gibco, 11140050), 2 mmol/L L-glutamine (Gibco, 35050061), 1% (v/v) nucleoside mix (Sigma-Aldrich, A-4036, T-1895, C-4654, G-6264, U-3003), 0.1 mmol/L β-mercaptoethanol (Sigma-Aldrich, M6250), 1,000 U/mL recombinant leukemia inhibitory factor (LIF) (Millipore, ESG1107).

Huang X., Wei C., Li F., Jia L., Zeng P., Li J., Tan J., Sun T., Jiang S., Wang J., Tang X., Zhao Q., Liu B., Rong L., Li C, & Ding J. (2019). PCGF6 regulates stem cell pluripotency as a transcription activator via super-enhancer dependent chromatin interactions. Protein & Cell, 10(10), 709-725.

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Photoreceptor Cell Culture and Gene Knockdown

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Cone photoreceptor cells (al-Ubaidi et al., 1992 (link); Tan et al., 2004 (link)) were cultured as monolayers at 37°C and 5% CO₂ in a humidified atmosphere in DMEM with FBS 10% supplemented with 20 µg/500 ml hydrocortisone (H-2270; Sigma-Aldrich), 20 µg/500 ml progesterone (P-8783; Sigma-Aldrich), 0.016 g/500 ml putrescine (P-7505; Sigma-Aldrich), and 20 µl/500 ml β-mercaptoethanol (M-6250; Sigma-Aldrich). No mycoplasma contamination of the cells was detected. An equal number of 661W cells (3 × 10⁵) was plated in each well of 6-well dishes. Cells were treated with AAV2-hRK-sh_control or AAV2-hRK–sh_Vldlr for overnight. On day 2, the cell medium was changed and treated with AAV2-hRK-sh_control or AAV2-hRK–sh_c-fos for overnight. On day 3, the medium was changed with fresh culture medium. After 48 h, photoreceptors were collected to determine gene expression by real time PCR.

Sun Y., Lin Z., Liu C.H., Gong Y., Liegl R., Fredrick T.W., Meng S.S., Burnim S.B., Wang Z., Akula J.D., Pu W.T., Chen J, & Smith L.E. (2017). Inflammatory signals from photoreceptor modulate pathological retinal angiogenesis via c-Fos. The Journal of Experimental Medicine, 214(6), 1753-1767.

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