Hydrocortisone h4001

Manufactured by Merck Group

Sourced in United States

Hydrocortisone (H4001) is a laboratory reagent used in various research applications. It is a naturally occurring steroid hormone with anti-inflammatory and immunosuppressive properties. This product is intended for research use only and its core function is to serve as a chemical compound for scientific investigation.

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10 protocols using hydrocortisone h4001

Visualizing HaloTagged AP-2 in Eps15 Knockout Cells

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Human-derived SUM159 cells gene-edited to add a HaloTag to both alleles of AP-2 σ2 were a gift from T. Kirchhausen^{54 (link)}. Cells were further gene-edited to knock out both alleles of endogenous Eps15 using CRISPR-associated protein 9 (Cas9) to produce the Eps15 knockout cells developed previously by our group^{12 (link)}.
Cells were grown in 1:1 DMEM high glucose: Ham’s F-12 (Hyclone, GE Healthcare) supplemented with 5% fetal bovine serum (Hyclone), Penicillin/Streptomycin/l-glutamine (Hyclone), 1 μg ml⁻¹ hydrocortisone (H4001; Sigma-Aldrich), 5 μg ml⁻¹ insulin (I6634; Sigma-Aldrich) and 10 mM HEPES, pH 7.4 and incubated at 37 °C with 5% CO₂. Cells were seeded onto acid-washed coverslips at a density of 3 × 10⁴ cells per coverslip for 24 h before transfection with 1μg of plasmid DNA using 3 μl Fugene HD transfection reagent (Promega). HaloTagged AP-2 σ2 was visualized by adding Janelia Fluor 646- HaloTag ligand (Promega). Ligand (100 nM) was added to cells and incubated at 37 °C for 15 min. Cells were washed with fresh medium and imaged immediately.

Yuan F., Gollapudi S., Day K.J., Ashby G., Sangani A., Malady B.T., Wang L., Lafer E.M., Huibregtse J.M, & Stachowiak J.C. (2023). Ubiquitin-driven protein condensation promotes clathrin-mediated endocytosis. bioRxiv.

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SUM159 Cell Culture and Stable Transfection

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Human-derived, mostly diploid SUM159 cells were grown in DMEM/F-12/GlutaMAX (10565-042; Thermo Fisher Scientific, Waltham, MA) supplemented with 5% fetal bovine serum (FBS; S11150; Atlanta Biologicals, Flowery Branch, GA), 100 U/ml penicillin and streptomycin (45000-652; VWR International, Radnor, PA), 1 μg/ml hydrocortisone (H4001; Sigma-Aldrich, St. Louis, MO), 5 μg/ml insulin (I9278; Sigma-Aldrich), and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; 25-060-CI; Mediatech, Manassas, VA), pH 7.4. The BSC1 monkey kidney epithelial cells stably expressing eGFP-CAAX (Boucrot and Kirchhausen, 2007 (link)) together with H2B-mCherry were generated by transfection with a plasmid encoding H2B-mCherry. Cells simultaneously expressing eGFP-CAAX and H2B-mCherry were selected for 7 d using 50 μg/ml puromycin, followed by cell sorting with FACSAria II (BD Biosciences, San Jose, CA). Cells were grown in DMEM (10569-044; Life Technologies) supplemented with 10% FBS, 100 U/ml penicillin and streptomycin, and, when required, 50 μg /ml puromycin.

Aguet F., Upadhyayula S., Gaudin R., Chou Y.Y., Cocucci E., He K., Chen B.C., Mosaliganti K., Pasham M., Skillern W., Legant W.R., Liu T.L., Findlay G., Marino E., Danuser G., Megason S., Betzig E, & Kirchhausen T. (2016). Membrane dynamics of dividing cells imaged by lattice light-sheet microscopy. Molecular Biology of the Cell, 27(22), 3418-3435.

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Culturing and Imaging of Breast Cancer Cells

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All cells were grown and imaged at 37°C and 5% CO_2. SUM159 cells were grown in DMEM/F12/Glutamax (Life Technologies, Grand Island, NY), supplemented with 5% fetal bovine serum (FBS), 100 U/ml penicillin and streptomycin (Life Technologies), 1 μg/ml hydrocortisone (H-4001; Sigma-Aldrich), 5 μg/ml insulin (128-100; Cell Applications, San Diego, CA), and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.4. Sum-Dyn2 cells stably expressing mCherry-LCa were obtained by transfection. hCLTA^EN/DNM2^EN cells were grown in DMEM/F12/Glutamax supplemented with 10% FBS and 100 U/ml penicillin and streptomycin. Cells (2 ml of medium containing 4 × 10⁴ cells) were placed on top of a coverslip in a six-well plate and imaged 12–24 h after plating.

Cocucci E., Gaudin R, & Kirchhausen T. (2014). Dynamin recruitment and membrane scission at the neck of a clathrin-coated pit. Molecular Biology of the Cell, 25(22), 3595-3609.

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Visualization of HaloTagged AP-2 σ2 in SUM159 Cells

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Human-derived SUM159 cells gene-edited to add HaloTag (Promega) to both alleles of AP-2 σ2 were a generous gift from Tom Kirchhausen^{29 (link)}. Cells were grown in 1:1 DMEM high glucose:Ham’s F-12 (Hyclone, GE Healthcare) supplemented with 5% fetal bovine serum (Hyclone), Pen/Strep/L-glutamine (Hyclone), 1 μg/ml hydrocortisone (H4001; Sigma-Aldrich), 5 μg/ml insulin (I6634; Sigma-Aldrich), and 10 mM HEPES (Fisher), pH 7.4 and incubated at 37°C with 5% CO₂. Cells were seeded onto acid-washed coverslips at a density of 3x10⁴ cells per coverslip for 24 h before transfection with 1-3 μg of plasmid DNA using 3 μL Fugene HD transfection reagent (Promega).
HaloTagged AP-2 σ2 was visualized by adding Janelia Fluor 646-HaloTag ligand, a gift from Luke Lavis³⁰. 100 nM ligand was added to cells and incubated at 37°C for 15 minutes. Cells were washed with fresh media and imaged immediately.

Day K.J., Kago G., Wang L., Richter J.B., Hayden C.C., Lafer E.M, & Stachowiak J.C. (2021). Liquid-like protein interactions catalyze assembly of endocytic vesicles. Nature cell biology, 23(4), 366-376.

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Plasma Cortisol and Oxytocin Measurements

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Plasma cortisol was measured with an extracted radioimmunoassay [17] (link) using hydrocortisone (H-4001; Sigma Chemical Company, St Louis, MO, USA) as standard. The assay used [3H]-cortisol (Amersham Pharmacia Biotech, UK, Buckinghamshire HP, England) as tracer and a dichloromethane extracion procedure with a mean (± standard error of the mean [SEM]) recovery of 93.2 ± 2.8%. There were 8 assays conducted, and the sensitivity ranged from 0.15 to 0.47 ng/mL with a mean of 0.33 ng/mL. The mean intra-assay coefficient of variation was 7.81%. The mean interassay coefficient of variation was 12.06%.
Oxytocin in plasma was measured using a Phoenix Pharmaceuticals Oxytocin Radioimmunoassay Kit (Belmont, CA, USA) following a similar procedure to that described by Marazziti [18] . There was a 100% cross-reactivity with oxytocin and no cross-reactivity with AVP. Nine assays were conducted and the sensitivity ranged from 0.3 to 2.3 pg/mL with a mean of 1.2 pg/mL. The mean intra-assay coefficient of variation was 5.22%, and the mean interassay coefficient of variation was 8.01%.

Ralph C.R, & Tilbrook A.J. (2016). The hypothalamo-pituitary-adrenal (HPA) axis in sheep is attenuated during lactation in response to psychosocial and predator stress. Domestic Animal Endocrinology, 55, 66-73.

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Serum Cortisol Measurement by RIA

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All samples were measured in a single assay. Serum cortisol concentrations were measured before and after 8 weeks of antidepressant treatment using an extracted RIA. RIA used hydrocortisone (H-4001, Sigma Chemical Company, St Louis, MO, USA) as a standard. The assay utilized ³H-cortisol (Amersham Pharmacia Biotech UK, Buckinghamshire HP, England) as tracer and a dichloromethane extraction procedure with a mean (± SEM) recovery of 91.2 ± 1.8%. All samples were measured in a single assay, which had a sensitivity of 0.54 ng/ml. The cross reactivity to chemicals that interfere with the assay result and their concentration (ug/dL) was checked. The cross reactivity of chemicals were: 11-Deoxycortisol (13.6%), cortisone (1.4%), dexamethasone (2.0%), corticosterone (8.0%), and prednisolone (32.2%).

Alenko A., Markos Y., Fikru C., Tadesse E, & Gedefaw L. (2020). Association of serum cortisol level with severity of depression and improvement in newly diagnosed patients with major depressive disorder in Jimma medical center, Southwest Ethiopia. PLoS ONE, 15(10), e0240668.

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Serum Cortisol Measurement Using RIA

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All samples were measured in a single assay. Serum cortisol concentrations were measured using an extracted radioimmunoassay (RIA). RIA used hydrocortisone (H-4001, Sigma Chemical Company, St Louis, MO, USA) as a standard. The assay utilized 3H-cortisol (Amersham Pharmacia Biotech UK, Buckinghamshire HP, England) as a tracer and a dichloromethane extraction procedure with a mean (±SEM) recovery of 91.2 ± 1.8%. The instrument had a sensitivity of 0.54 ng/ml. The cross-reactivity to chemicals that interfere with the assay result and their concentration (μg/dL) was checked. The cross-reactivity of chemicals were: 11-Deoxycortisol (13.6%), cortisone (1.4%), dexamethasone (2.0%), corticosterone (8.0%), and prednisolone (32.2%).

Woldesenbet Y.M., Alenko A., Bukata I.T., Gedefaw L, & Fikru C. (2021). The status of serum cortisol before and after treatment of schizophrenia and its correlation to disease severity and improvement: A longitudinal study. SAGE Open Medicine, 9, 20503121211056216.

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Visualization of HaloTagged AP-2 σ2 in SUM159 Cells

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Culturing Primary Epithelial Cells from Surgical Tissues

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Cultures of primary, untransformed epithelial cells were generated from ex-vivo tissues, discarded following routine vaginal repair surgeries or hysterectomies, and cultured as described^{30 –32 (link)}. Cells were maintained in F medium (3:1 [v/v] F12 [Ham]-DMEM [Life Technologies], 5% fetal calf serum [Gemini Bio- Products], 0.4 μg/ml hydrocortisone [H-4001; Sigma], 5 μg/ml insulin [700-112 P; Gemini Bio-Products], 8.4 ng/ml cholera toxin [227036; EMD Millipore], 10 ng/ml epidermal growth factor [PHG0311; Life Technologies], 24 μg/ml adenine [A-2786; Sigma], 100 U/ml penicillin, and 100 μg/ml streptomycin [Life Technologies]). Cells were routinely cultured in the presence of irradiated (6000 Rad) 3T3-J2 feeder fibroblasts and 10 μM of Rho kinase inhibitor Y27632 (1254; Enzo Life Sciences). Feeder cells were detached first by 1 min treatment with 10 ml Versene (Life Technologies), followed by 5 min treatment with trypsin/EDTA (Life Technologies) to dislodge epithelial cells. 100,000 epithelial cells were plated in each well in 1 ml of medium in 12 well plates 1 day prior to infection.

Fink S.L., Vojtech L., Wagoner J., Slivinski N.S., Jackson K.J., Wang R., Khadka S., Luthra P., Basler C.F, & Polyak S.J. (2018). The Antiviral Drug Arbidol Inhibits Zika Virus. Scientific Reports, 8, 8989.

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Enzyme-Linked Immunosorbent Assay for Cortisol Detection

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A Cyclopore polycarbonate (PC) membrane, containing 5 µm diameter conical-shaped micropores (Catalog No 110613; Whatman, Maidstone, U.K.), was employed as the template. Hydrocortisone (H4001, MW 362.46 g mol -1 ), Sodium dodecyl sulfate (SDS, MW 288.38 g mol -1 ), 3,4-ethylenedioxythiophene (EDOT), potassium nitrate (KNO 3 ), hydrogen peroxide (H 2 O 2 ), 11-Mercaptoundecanoic acid (MUA), 6-Mercapto-1-hexanol (MCH), 4-morpholineethanesulfonic acid hemisodium salt (MES), 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC), and N-hydroxylsulfosuccinimide (Sulfo-NHS) were purchased from Sigma-Aldrich ® . The monoclonal cortisol antibody (Catalog # P01-92-94 M-P) and cortisol-HRP antigen (Catalog # P91-92-91H) were purchased from EastCoast Bio®. K-Blue® Aqueous 3,3′,5,5′-tetramethylbenzidine (TMB) substrate was purchased from Neogen® Corporation.
The antibody was dissolved in 0.05 M phosphate buffer (PBS) pH 7.4. The washing solution consisted of 0.05 M phosphate buffer pH 7.4 containing 0.05% Tween-20.
The aqueous hydrogen peroxide solutions were used as the chemical fuel, and Sodium dodecyl sulfate (SDS, MW 288.38 g mol -1 ) (at 3% final concentration) was used as a surfactant in all propulsion experiments.
Ultrapure water (18MX cm, Millipore Corporation, USA) was used for the preparation of all aqueous solutions.

de Ávila B.E., Zhao M., Campuzano S., Ricci F., Pingarrón J.M., Mascini M, & Wang J. (2017). Rapid micromotor-based naked-eye immunoassay. Talanta, 167.

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