M8046

Manufactured by Merck Group

Sourced in United States

The M8046 is a laboratory equipment product manufactured by Merck Group. It is designed for general laboratory use, performing core functions essential to various research and analysis applications. The product specifications and technical details are available upon request.

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Lab products found in correlation

Mifepristone, by Merck Group (4 mentions) D1756, by Merck Group (3 mentions) Ru486, by Merck Group (3 mentions) Penicillin and streptomycin, by Thermo Fisher Scientific (2 mentions) Dmem f12 media, by Thermo Fisher Scientific (2 mentions) Whatman polycarbonate membranes, by Thermo Fisher Scientific (2 mentions) P3755, by Merck Group (2 mentions) M8506, by Merck Group (2 mentions) D2915, by Merck Group (2 mentions) Spironolactone, by Merck Group (2 mentions) Dexamethasone (dex), by Merck Group (2 mentions) Eplerenone, by Tokyo Chemical Industry (2 mentions) Dapi dilactate, by Thermo Fisher Scientific (1 mentions) Ab143597, by Abcam (1 mentions) Dmi6000b, by Leica camera (1 mentions) Ab150077, by Abcam (1 mentions) Norvir, by Abbvie (1 mentions) Pure ethanol, by Merck Group (1 mentions) Bafa1, by Merck Group (1 mentions) M9281, by Merck Group (1 mentions)

17 protocols using m8046

Corticosterone and GR Antagonist Effects on Caco-2 and HepG2 Cells

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Freshly thawed Caco-2 cells or HepG2 cells were cultured in DMEM + 10% fetal bovine serum medium at 37 °C at 5% CO₂. When cell confluence reached 80%, cells were subcultured in a 6-well/24-well plate and incubated for 24 h, then the cells were treated with the following reagents: Corticosterone (1 μM or 10 μM, ab143597, Abcam, MA, USA), and/or RU486 (10 μM, M8046, Sigma-Aldrich, St. Louis, MO, USA), a GR antagonist. After 12 h or 24 h of treatment, the cells were collected to extract RNA and protein or immunofluorescence.
Caco-2 cells or HepG2 cells were fixed in 4% neutral paraformaldehyde for 20 min in a shaker. PBS containing 0.5% TritonX-100 was added to break the membrane at room temperature for 20 min, blocked with 5% FBS, and then incubated with rabbit anti-GR antibody (24050-1-AP, Proteintech., Chicago, IL, USA) overnight at 4 °C and goat anti-rabbit IgG (ab150077, Abcam, MA, USA) for 1 h. DAPI (DAPI dilactate, Cat #D3571, Invitrogen, CA, USA) was used to stain the cell nuclei. Subsequently, the samples were viewed with a fluorescence microscope (Leica, DMI6000 B, Germany).

Guo S., Chen Z., Dong Y., Ni Y., Zhao R, & Ma W. (2023). Chronic Corticosterone Exposure Suppresses Copper Transport through GR-Mediated Intestinal CTR1 Pathway in Mice. Biology, 12(2), 197.

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Mifepristone and Ritonavir Pharmacokinetics

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Mfp was obtained as pure substance from Sigma (M8046) or as tablet formulation Mifegyne (200 mg/tablet; Mfp content confirmed by high-performance liquid chromatography [HPLC] with diode array detection, see below) from Nordic Pharma. Rtv was obtained from AbbVie as Norvir 100 mg. Mfp and Rtv were either diluted in DMSO for i.p. application or suspended in ASV (0.9% NaCl [w/v], 0.5% carboxymethyl-cellulose MW 250.000 [w/v], 0.4% polysorbate 80 [v/v], 0.9% benzyl alcohol [v/v]). Mfp was also used as suspension in sesame oil.
Drug dosages applied to rats and monkeys can be converted to HEDs according to the FDA-approved body surface area (BSA) normalization approach.⁴⁸ ^,⁴⁹ (link) Specifically, calculating

H E D (m g / k g) = a n i m a l d o s e (m g / k g) x {(a n i m a l w e i g h t k g / h u m a n w e i g h t k g)}^{1 - 0.67}

results in the following conversion factors: for rats weighing 250 g, monkeys weighing 3 kg, and an assumed human weight of 70 kg: rat dosage (e.g., 50 mg/kg) × 0.156 = HED (7.8 mg/kg); monkey dosage (e.g., 50 mg/kg) × 0.357 = HED (17.9 mg/kg). In addition, it must be considered that bioavailability of Mfp in monkeys (15%) is considerably lower compared to humans and rats (40%),¹⁹ justifying higher dosages to be applied in monkeys as compared to rats.

Cheng S., van Gaalen M.M., Bähr M., Garea-Rodriguez E, & Kügler S. (2021). Optimized pharmacological control over the AAV-Gene-Switch vector for regulable gene therapy. Molecular Therapy. Methods & Clinical Development, 23, 1-10.

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Inducing Conditional Gene Expression in Flies

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Flies were cultured at 25 °C before RU486 treatment and maintained as described above; at the desired age, flies were switched to vials containing normal diet plus a wet yeast paste mixed with RU486 (M8046, Sigma). A 10 mg/mL stock solution of RU486 (mifepristone; Sigma) was made in pure ethanol (Sigma). For RU486-containing wet yeast preparation, 0.28 mL of RU486 stock solution, 0.1% blue food color additive (to confirm food intake; #861146, Sigma) and 1.52 mL ddH₂O were mixed well and then added to 1 g active dry yeast (RED STAR) for a final concentration of 3.6 mM RU486.
For 3TC (2′, 3′-Dideoxy-3′-thiacytidine, #L1295, Sigma) treatment, newly eclosed flies were cultured in plastic vials containing normal diet plus a 3TC-containing wet yeast paste. For 3TC-containing wet yeast preparation, 11.5 mg 3TC, 0.1% blue food color and 3.3 mL ddH₂O was mixed well and then added to 1.7 g active dry yeast for a final concentration of 10 mM 3TC. Control flies were fed with the same diet but without RU486 or 3TC. Food was changed daily until flies were dissected.

Lin K.Y., Wang W.D., Lin C.H., Rastegari E., Su Y.H., Chang Y.T., Liao Y.F., Chang Y.C., Pi H., Yu B.Y., Chen S.H., Lin C.Y., Lu M.Y., Su T.Y., Tzou F.Y., Chan C.C, & Hsu H.J. (2020). Piwi reduction in the aged niche eliminates germline stem cells via Toll-GSK3 signaling. Nature Communications, 11, 3147.

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Culturing Lung Slices for BADJ Formation

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E13 lungs or E14 lung slices of 200~300 um thickness were isolated in ice-cold PBS and cultured on Whatman polycarbonate membranes (09-300-53, Fisher Scientific) in DMEM/F12 media (11320032, Invitrogen) with 1% fetal bovine serum (16140063, Invitrogen) and penicillin and streptomycin (15140148, Invitrogen). To activate Sftpc-rtTA, 2 ug mL⁻¹ doxycycline was added to the culture media. To screen for hormonal regulators of BADJ formation, the following chemicals were added to the culture media: dexamethasone (100 nM, D2915, Sigma), 6-propyl-2-thiouracil (100 uM, P3755, Sigma), methimazole (50 uM, M8506, Sigma), and acitretin (10 uM, 44707, Sigma), mifepristone (3 uM, M8046, Sigma). Cultured lungs were imaged daily on a fluorescence stereomicroscope (M205C, Leica).

Alanis D.M., Chang D.R., Akiyama H., Krasnow M.A, & Chen J. (2014). Two Nested Developmental Waves Demarcate a Compartment Boundary in the Mouse Lung. Nature communications, 5, 3923.

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Assessing Corticoid Receptor Specificity

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To assess the target specificity of the glucocorticoid and mineralocorticoid stimulation, we used the inhibitors mifepristone (M8046, Sigma-Aldrich) and canrenone (SML 1497, Sigma-Aldrich), respectively. Cells were seeded at 2 × 10³ cells cm⁻² for the specified duration of experiments (7 days for the metabolomics experiments and 3 weeks to assess the long-term effect of mifepristone-induced glucocorticoid inhibition on osteogenic marker expression). Mifepristone was used at 10 μM, and canrenone was used at 100 μM, and they were supplemented in the medium with every feed (twice per week) for the duration of each experiment.

Hodgkinson T., Tsimbouri P.M., Llopis-Hernandez V., Campsie P., Scurr D., Childs P.G., Phillips D., Donnelly S., Wells J.A., O’Brien F.J., Salmeron-Sanchez M., Burgess K., Alexander M., Vassalli M., Oreffo R.O., Reid S., France D.J, & Dalby M.J. (2021). The use of nanovibration to discover specific and potent bioactive metabolites that stimulate osteogenic differentiation in mesenchymal stem cells. Science Advances, 7(9), eabb7921.

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Culturing Lung Slices for BADJ Formation

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Alanis D.M., Chang D.R., Akiyama H., Krasnow M.A, & Chen J. (2014). Two Nested Developmental Waves Demarcate a Compartment Boundary in the Mouse Lung. Nature communications, 5, 3923.

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Cell Culture and Inhibitor Treatments

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Mouse hepatoma Hepa1-6 cells, human cervical carcinoma HeLa cells, human hepatoma HepG2 cells, and human lung carcinoma A549 cells were purchased from the ATCC. The cells were cultured in DMEM (25 mM glucose, 11995040, Gibco) supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and 1% HEPES (BOSTER, PYG0019) at 37 °C and 5% CO₂. GFP-LC3/HeLa cells were maintained in DMEM (25 mM glucose), and supplemented with 0.4 mg/mL G418 (10131035, Thermo Scientific, Waltham, MA, USA) at 37 °C and 5% CO₂.
For inhibitory assays, the antagonists or inhibitors, such as RU486 (30 μM, M8046, Sigma-Aldrich, St. Louis, MO, USA), were used to treat Hepa1-6 cells for 12 h; similarly, BafA1 (100 nM, B1793, Sigma-Aldrich) and 3-MA (10 mM, M9281, Sigma-Aldrich) were used to treat GFP-LC3/HeLa cells or Hepa1-6 cells for indicated time.

Yang S., Liu T., Hu C., Li W., Meng Y., Li H., Song C., He C., Zhou Y, & Fan Y. (2022). Ginsenoside Compound K Protects against Obesity through Pharmacological Targeting of Glucocorticoid Receptor to Activate Lipophagy and Lipid Metabolism. Pharmaceutics, 14(6), 1192.

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Glucocorticoid and Mineralocorticoid Receptor Modulation in Zebrafish

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For glucocorticoid and mineralocorticoid receptor agonist and antagonist treatment, dexamethasone (D1756, Sigma-Aldrich), spironolactone (S3378, Sigma-Aldrich), RU486 (Mifepristone, M8046, Sigma-Aldrich) and eplerenone (E0905, TCI chemicals) were dissolved in DMSO to make stock solutions. Uninjured adult fish were incubated in 350 μM dexamethasone or 25 μM spironolactone or DMSO as vehicle control. Fresh drug water was changed every 24 h for 4 days. At 4 days post treatment, heart, brain and kidney were collected for histology analysis. Z-CAT fish treated with tamoxifen were incubated in 5 μM RU486 or 50 μM eplerenone or DMSO as vehicle control from 3 days post tamoxifen treatment to 6 days post tamoxifen treatment. Water was changed every 24 h. At 7 days post tamoxifen treatment (4 days post drug treatment), the brain was collected for quantitative PCR.

Sun F., Ou J., Shoffner A.R., Luan Y., Yang H., Song L., Safi A., Cao J., Yue F., Crawford G.E, & Poss K.D. (2022). Enhancer selection dictates gene expression responses in remote organs during tissue regeneration. Nature cell biology, 24(5), 685-696.

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Cytokine Production Assay with TA

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Cytokine production was determined using 96-well cell culture supernatants and commercial ELISA kits for TNF-a (R&D Systems) and IL-6 (Sanquin) following the instructions of the manufacturer. One hour before LPS treatment on day 6 of Mo differentiation^{43 (link)}, the medium was refreshed and 1 nM to 1 μM TA and optionally 1 μM RU486 of the GR antagonist (Sigma, M8046) were added to the medium. LPS (100 ng ml⁻¹) treatment lasted 24 h at which point the supernatant was collected for ELISA.

Wang C., Nanni L., Novakovic B., Megchelenbrink W., Kuznetsova T., Stunnenberg H.G., Ceri S, & Logie C. (2019). Extensive epigenomic integration of the glucocorticoid response in primary human monocytes and in vitro derived macrophages. Scientific Reports, 9, 2772.

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Modeling In Vivo Stress Response

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For in vivo experiments, mifepristone (RU486; 50 mg/kg, Sigma, caltalog M8046), cort (1.5 mg/kg, Sigma, catalog C174), and dexamethasone (dex; 2 mg/kg, Sigma, catalog D1756) were prepared in a sterile peanut oil vehicle (veh) and then injected in a 0.1-ml volume 1 h before cell harvest. Cells were plated and kept in Neurobasal A media for 72 h. The dose of injected cort was previously determined to reproduce stress-induced plasma cort concentrations (Alexander et al., 2009 (link)). All drugs were prepared fresh daily and delivered via intraperitoneal injection.

Lerch J.K., Alexander J.K., Madalena K.M., Motti D., Quach T., Dhamija A., Zha A., Gensel J.C., Webster Marketon J., Lemmon V.P., Bixby J.L, & Popovich P.G. (2017). Stress Increases Peripheral Axon Growth and Regeneration through Glucocorticoid Receptor-Dependent Transcriptional Programs. eNeuro, 4(4), ENEURO.0246-17.2017.

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