Insulin

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Insulin is a laboratory product used for research and experimental purposes. It is a hormone produced by the pancreas that regulates blood sugar levels. Insulin plays a key role in the metabolism of carbohydrates, fats, and proteins.

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Bovine insulin (5 citations) Insulin receptor phosphorylated on Tyr960 (2 citations)

10 protocols using insulin

Culturing Primary Dermal Fibroblasts and SUM159 Cells

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Primary dermal fibroblasts from both patients were provided by the Manton Center for Orphan Disease Research at Boston Children’s Hospital. Primary dermal fibroblasts from three control subjects were provided by the Memory and Aging Center at the University of California, San Francisco; the control cell lines used were CTR2 and V, as described (21 (link), 22 (link)), and JJ (unpublished). Primary human dermal fibroblasts were cultured in Ham’s F-10 nutrient mix (Thermo Fisher Scientific) containing FBS (10%), penicillin (100 U/ml), and streptomycin (100 mg/ml).
Human breast cancer cells (SUM159 cells) were maintained in DMEM/F-12 GlutaMAX (Life Technologies, Carlsbad, CA) containing FBS (5%), penicillin (100 U/ml), streptomycin (100 mg/ml), hydrocortisone (1 mg/ml) (Sigma-Aldrich, St. Louis, MO), insulin (5 mg/ml) (Cell Applications, San Diego, CA), and HEPES (10 mM; pH 7.0). Transfection of plasmid was performed using Lipofectamine 3000 (Life Technologies) according to the manufacturer’s instructions.

Gluchowski N.L., Chitraju C., Picoraro J.A., Mejhert N., Pinto S., Xin W., Kamin D.S., Winter H.S., Chung W.K., Walther T.C, & Farese RV J.r. (2017). Identification and characterization of a novel DGAT1 missense mutation associated with congenital diarrhea. Journal of Lipid Research, 58(6), 1230-1237.

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Synchronized Mineralization of HeLa and Sum159 Cells

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Hela cells were cultured in DMEM (Thermo Fisher Scientific), supplemented with 10% (v/v) Fetal Bovine Serum (FBS; Biochrom) and 100 mg/mL penicillin-streptomycin (Thermo Fisher Scientific).
Sum159 cells were cultured in DMEM/F12, glutaMAX media (Life Technology) supplemented with 5% FBS, 1 μg/ml hydrocortisone (Sigma), 5 ug/ml Insulin (Cell Applications), 10 mM HEPES (pH 7.4), and 100 mg/ml penicillin-streptomycin (Thermo Fisher Scientific). Cells were maintained in TC-25 flasks (Thermo Fisher Scientific) at 37 °C under 5% CO2. For EM grid seeding, cells at 60-70% confluence were treated with 0.05% trypsin-EDTA (Fisher Scientific) for 2 min at 37 ) for 12 h. Then Ca 2+ was supplemented to the normal seawater level of 10 mM. This lag time in a calcium depleted medium allowed the cells to resume mineralization in a synchronized fashion upon Ca 2+ repletion. Cells were centrifuged 3 h after re-calcification at 2000 g for 3 min to increase their concentration for plunge freezing. A volume of 4 µL cell suspension at a concentration of 3.07-3.5x10 7 cells/ml was directly applied to glow-discharged holey carbon R2/1 Cu 200 mesh grids (Quantifoil). After 5 s back-side blotting, plunge-freezing was carried out with a Leica GP EM at 18°C and 90% humidity.

Klumpe S., Fung H.K., Goetz S.K., Zagoriy I., Hampoelz B., Zhang X., Erdmann P.S., Baumbach J., Müller C.W., Beck M., Plitzko J.M., & Mahamid J. (2021). A Modular Platform for Streamlining Automated Cryo-FIB Workflows.

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Reagents for Cell Signaling Experiments

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AVE0991 was a kind gift from Sanofi‐Aventis (Frankfurt, Germany); A799 was from Bachem (Bubendorf, Switzerland); IFN‐γ and TNF‐α were supplied by PeproTech EC (London, UK); insulin was from Cell Applications (San Diego, CA) and PGF_2α was supplied by Cayman Chemical Company (Ann Arbor, MI). Sigma‐Aldrich (St Louis, MO) supplied ACh, LPS (from

Escherichia coli

0111:B4;), dexamethasone, IBMX and SNP.

Skiba D.S., Nosalski R., Mikolajczyk T.P., Siedlinski M., Rios F.J., Montezano A.C., Jawien J., Olszanecki R., Korbut R., Czesnikiewicz‐Guzik M., Touyz R.M, & Guzik T.J. (2017). Anti‐atherosclerotic effect of the angiotensin 1–7 mimetic AVE0991 is mediated by inhibition of perivascular and plaque inflammation in early atherosclerosis. British Journal of Pharmacology, 174(22), 4055-4069.

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Cell Culture and Fatty Acid Treatments

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K562 cells (ATCC CCL-243) were cultured in flasks using RPMI medium (Gibco) containing 25 mM Hepes and 2 mM glutamine. The medium was supplemented with 2 mM glutamine (Gibco), 10% FBS (Thermo) and Pen/Strep (Gibco). Cells were kept in active proliferation (max 100*10⁴/ml) for the duration of the experiments. Cells reaching 100*10⁴/ml were diluted 1/4 or 1/8 using fresh medium. Standard growing conditions were used (i.e., 37°C and 95% humidity). Cells were treated with palmitate or palmitoleate as indicated in figure legends. In general, high palmitate concentration (e.g., 0.2–0.25 mM for 24–28 h) were used for viability assays. Lower concentration (e.g., 0.15–0.2 mM for 16–20h) were used in all other experiments (including western blotting, RNAsq and lipidomics) to avoid carryover of more than 5–10% of death cells.
SUM159 cells (RRID: CVCL_5423, obtained from the lab of Tomas Kirchhausen) were maintained in DMEM/F-12 GlutaMAX (Life Technologies) with 5 ug/ml insulin (Cell Applications), 1 ug/ml 5% FBS (Life Technologies), 50 U/ml streptomycin and 50 U/ml penicillin. Hydrocortisone (Sigma), HepG2 cells (ATCC) cand HeLa cells (obtained from Thomas Kirchhausen) were cultured in DMEM supplemented with 10% FBS (Life Technologies) and 50 U/ml streptomycin and 50 U/ml penicillin.

Piccolis M., Bond L.M., Kampmann M., Pulimeno P., Chitraju C., Jayson C.B., Vaites L.P., Boland S., Lai Z.W., Gabriel K.R., Elliott S.D., Paulo J.A., Harper J.W., Weissman J.S., Walther T.C, & Farese RV J.r. (2019). Probing the Global Cellular Responses to Lipotoxicity Caused by Saturated Fatty Acids. Molecular cell, 74(1), 32-44.e8.

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Cytochrome c Enzymatic Assays in Cells

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Cytochrome c (cyt c) (horse heart), glucose-6-phosphate (G6P), glucose-6-phosphate dehydrogenase (G6PD), nicotinamide-adenine dinucleotide phosphate (NADPH), ethoxy resorufin, methoxy resorufin, resorufin, coumarin, 7-hydroxy coumarin, 3-cyano-7-ethoxycoumarin, 3-cyano-7-hydroxycoumarin, fluorescein, trypsin, penicillin-streptomycin (10,000 units penicillin and 10 mg streptomycin per mL), Dulbecco’s Modified Eagle’s Medium-low glucose (DMEM), fetal bovine serum (FBS), phosphate buffered saline pH 7.4 (PBS) and doxorubicin were obtained from Sigma Aldrich (St. Louis, MO, USA). Nicotinamide adenine dinucleotide phosphate (NADP+) was obtained from Gerbu (Heidelberg, Germany). Bradford reagent was obtained from Bio-Rad (Hercules, CA, USA) and Quiazol from Qiagen (Hilden, Germany). Dibenzylfluorescein, sodium dithionite, dimethyl sulfoxide (DMSO), acetonitrile (ACN) and sodium hydrogencarbonate (NaHCO₃) were purchased from Merck (Kenilworth, NJ, USA). Insulin was obtained from Cell Applications, Inc. (San Diego, CA, USA). All other chemicals and solvents were of the highest grade commercially available.

Barata I.S., Gomes B.C., Rodrigues A.S., Rueff J., Kranendonk M, & Esteves F. (2022). The Complex Dynamic of Phase I Drug Metabolism in the Early Stages of Doxorubicin Resistance in Breast Cancer Cells. Genes, 13(11), 1977.

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Maintenance and Transfection of SUM159 Cells

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Human SUM159 cells (breast carcinoma cell line) were maintained in DMEM/F-12 GlutaMAX medium (Life Technologies) containing 5% FBS, 100 U/ml penicillin, 100 mg/ml streptomycin, 1 mg/ml hydrocortisone (MilliporeSigma), 5 mg/ml insulin (Cell Applications), and 10 mM Hepes, pH 7.0. Transfection of plasmids was performed 24 h before experiments with FuGENE-HD (Promega) according to the manufacturer’s instructions. siRNA treatment was performed 72 h before the experiment by reverse transfection using Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the manufacturer’s instructions. Human Expi293F GnTI− cells were grown in suspension in FreeStyle 293 medium (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Becuwe M., Bond L.M., Pinto A.F., Boland S., Mejhert N., Elliott S.D., Cicconet M., Graham M.M., Liu X.N., Ilkayeva O., Saghatelian A., Walther T.C, & Farese RV J.r. (2020). FIT2 is an acyl–coenzyme A diphosphatase crucial for endoplasmic reticulum homeostasis. The Journal of Cell Biology, 219(10), e202006111.

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Membrane Topology of MBOAT7 Constructs

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Membrane topology of MBOAT7 constructs was determined by fluorescence protease protection assay, as described previously^{34 (link)}. In brief, MBOAT7 and control (mCherry-ER3 and mEmerald-Sec61β) constructs was transfected into the SUM159 cells using the FuGENE® Transfection Reagent according to the manufacturer’s protocol. Two days post transfection, SUM159 cells were washed three times with 2 ml of KHM buffer (110 mM potassium acetate, 20 mM HEPES-KOH pH 7.2, 2 mM MgCl₂) at room temperature. During the imaging acquisition, 40 μM digitonin and 100 μg/ml proteinase K (Sigma) were added to the media at the indicated time points for plasma membrane permeabilization and fluorescent protein degradation, respectively. SUM159 breast cancer cells were obtained from the laboratory of Tomas Kirchhausen at Harvard Medical School (ATCC, CRL-1504) and were maintained in DMEM/F-12 GlutaMAX (Life Technologies) with 5 µg/ml insulin (Cell Applications), 1 µg/mL of hydrocortisone (Sigma), 5% FBS (Life Technologies 10082147, Thermo Fisher), 50 µg/mL of streptomycin, and 50 U/mL of penicillin.

Wang K., Lee C.W., Sui X., Kim S., Wang S., Higgs A.B., Baublis A.J., Voth G.A., Liao M., Walther T.C, & Farese RV J.r. (2023). The structure of phosphatidylinositol remodeling MBOAT7 reveals its catalytic mechanism and enables inhibitor identification. Nature Communications, 14, 3533.

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Maintenance of SUM159 Breast Cancer Cells

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SUM159 breast cancer cells were obtained from the laboratory of Tomas Kirchhausen (Harvard Medical School) and were maintained in Dulbecco’s modified Eagle medium (DMEM)/F12 GlutaMAX (Life Technologies) supplemented with 5 μg ml⁻¹ insulin (Cell Applications), 1 μg ml⁻¹ hydrocortisone (Sigma), 5% foetal bovine serum (Life Technologies 10082147; Thermo Fisher), 50 μg ml⁻¹ streptomycin and 50 U ml⁻¹ penicillin. Where noted, cells were incubated with medium containing 0.5 mM OA complexed with essentially fatty-acid-free bovine serum albumin (BSA). For immunoprecipitation analyses, HEK293T cells (ATCC) were used. HEK293T cells were cultured in DMEM (Gibco) supplemented with 10% foetal bovine serum (Life Technologies), 50 μg ml⁻¹ streptomycin and 50 U ml⁻¹ penicillin.

Chung J., Park J., Lai Z.W., Lambert T.J., Richards R.C., Zhang J., Walther T.C, & Farese RV J.r. (2023). The Troyer syndrome protein spartin mediates selective autophagy of lipid droplets. Nature Cell Biology, 25(8), 1101-1110.

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Culturing SUM159 Breast Cancer Cells

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SUM159 breast cancer cells (RRID:CVCL_5423) were obtained from the laboratory of Dr. Tomas Kirchhausen (Harvard Medical School) and were maintained in DMEM/F-12 GlutaMAX (Life Technologies) supplemented with 5 μg/ml insulin (Cell Applications), 1 μg/ml hydrocortisone (Sigma), 5% FBS (Life Technologies 10082147; Thermo Fisher), 50 μg/ml streptomycin, and 50 U/ml penicillin. Cell lines were tested monthly for mycoplasma contamination using the PCR Mycoplasma Test Kit (ABM Cat# G238) and always came back negative. The cell lines were authenticated via STR profiling.

Kim S., Chung J., Arlt H., Pak A.J., Farese RV J.n.r., Walther T.C, & Voth G.A. (2022). Seipin transmembrane segments critically function in triglyceride nucleation and lipid droplet budding from the membrane. eLife, 11, e75808.

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Culturing SUM159 Breast Cancer Cells

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SUM159 breast cancer cells were obtained from the laboratory of Tomas Kirchhausen (Harvard Medical School) and were maintained in DMEM/F-12 GlutaMAX (Life Technologies, #10565042) supplemented with 5 μg/ml insulin (Cell Applications), 1 μg/ml hydrocortisone (Sigma), 5% FBS (Life Technologies 10082147; Thermo Fisher), 50 μg/ml streptomycin, and 50 U/ml penicillin. Where noted, cells were incubated with media containing 100–500 μM oleic acid complexed with essentially fatty acid–free BSA. For protein purification and cryo-EM analysis, Expi293 suspension culture cells (Life Technologies) were used. Expi293 cells were cultured in Expi293 Expression Medium (#A1435102, Gibco) at 37°C under 8% CO₂ and 80% humidity in Multitron-Pro shaker at 125 rpm. To block TG synthesis, DGAT1 (Liu et al., 2013 (link)) and DGAT2 inhibitors (Imbriglio et al., 2015 (link)) from Merck & Co. were dissolved in DMSO (D2650, Sigma-Aldrich) and used at a final concentration as noted in the figure legends.

Chung J., Wu X., Lambert T.J., Lai Z.W., Walther T.C, & Farese RV J.r. (2019). Lipid droplet assembly factor-1 and seipin form a lipid droplet assembly complex. Developmental cell, 51(5), 551-563.e7.

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