Hepes

Manufactured by Americanbio

HEPES is a chemical buffer used in cell culture and biochemical applications. It helps maintain a stable pH environment for biological processes.

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Lab products found in correlation

Its supplement, by Corning (2 mentions) Glutamax, by Americanbio (2 mentions) Hi fbs, by Americanbio (2 mentions) Zinc sulfate, by Merck Group (2 mentions) Pen strep, by Thermo Fisher Scientific (2 mentions) Fluozin 3, by Thermo Fisher Scientific (2 mentions) Trypsin edta, by Thermo Fisher Scientific (2 mentions) Tnf α, by R&D Systems (2 mentions) Ifn γ, by R&D Systems (2 mentions) Facsaria 2, by BD (2 mentions) Sodium pyruvate, by Thermo Fisher Scientific (2 mentions) Fetal bovine serum (fbs), by Merck Group (2 mentions) L glutamine, by Merck Group (2 mentions) Complete protease inhibitor mixture tablets, by Merck Group (1 mentions) Imidazole, by Thermo Fisher Scientific (1 mentions) Mgcl2, by Merck Group (1 mentions) Dithiothreitol (dtt), by Thermo Fisher Scientific (1 mentions) Adenosine triphosphate (atp), by Merck Group (1 mentions) γ 32p atp, by PerkinElmer (1 mentions) β mercaptoethanol, by MP Biomedicals (1 mentions)

7 protocols using hepes

Human and Rat Islet Isolation and Culture

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Human islets from normal donors were purchased from Prodo Laboratories Inc. and were cultured immediately upon arrival in CMRL complete media consisting of glutamine free CMRL supplemented with 10 mM niacinamide and 16.7 uM zinc sulfate (Sigma), 1% ITS supplement (Corning), 5 mM sodium pyruvate, 1% Glutamax, 25 mM HEPES (American Bio), 10% HI FBS and antibiotics (10,000 units mL⁻¹ penicillin and 10 mg mL⁻¹ streptomycin). All media components were obtained from Life Technologies unless otherwise indicated. Human donor islets were cultured as intact islets then dispersed and re-aggregated as pseudo-islets for dynamic insulin secretion studies. The formation of single pseudo-islet aggregates were performed by first dispersing the intact islets using accutase (Gibco), then the resulting cell suspension was seeded at 5000 cells per well of a 96-well V-bottom plate, lightly centrifuged (~200×g) and then incubated for 12–24 h at 37 °C 5% CO₂/95%.
Rat islets were isolated from Sprague-Dawley rats and recovered in culture at 37 °C 5% CO₂/95% for 12–24 h in 5 mM glucose RPMI (Sigma) supplemented with 10 mM HEPES (American Bio), 10% FBS (Gemini Bio-products) and antibiotics (Life Technologies). The islets were then dispersed and re-aggregated into pseudo islets as described above.

Perry R.J., Rabin-Court A., Song J.D., Cardone R.L., Wang Y., Kibbey R.G, & Shulman G.I. (2019). Dehydration and insulinopenia are necessary and sufficient for euglycemic ketoacidosis in SGLT2 inhibitor-treated rats. Nature Communications, 10, 548.

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Culturing and Injecting Melanoma, OVA, and YUMMER1.7 Cells

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B16-F10 melanoma cells were cultured in DMEM and E.G7-OVA cells were cultured in RPMI1640, both were supplemented with 10% FBS (GIBCO), 1% penicillin-streptomycin (GIBCO), 2 mM L-glutamine (GIBCO), 1 mM sodium pyruvate (GIBCO), and 0.01 M HEPES (AmericanBio). YUMMER1.7 cells were provided by Dr. Marcus W. Bosenberg. YUMMER1.7 cells were cultured in DMEM/F-12 Media with 10% FBS (GIBCO), 1% penicillin-streptomycin (GIBCO), 2 mM L-glutamine (GIBCO), 1 mM sodium pyruvate (GIBCO), and 0.01 M HEPES (AmericanBio). For the in vivo injection, cells were diluted in OPTI-MEM with the proper concentration of injection.

Hong J.Y., Lim J., Carvalho F., Cho J.Y., Vaidyanathan B., Yu S., Annicelli C., Eddie W.K, & Medzhitov R. (2020). Long-term programming of CD8 T cell immunity by perinatal exposure to glucocorticoids. Cell, 180(5), 847-861.e15.

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Molecular Interactions in Tyrosine Kinase Signaling

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Primers were purchased from IDT (Coralville, IA) and used without further purification. HEPES was purchased from American Bio (Canton MA). β-mercaptoethanol was purchased from MP Biomedicals (Santa Ana, CA). Poly(6:2:5:1 Ala, Glu, Lys, Tyr) hydrobromide peptide, ATP, MgCl₂, complete protease inhibitor mixture tablets, and nickel-NTA chromatography resin were purchased from Sigma Aldrich (St. Louis, MO). DTT, NaCI, imidazole, Triton X-10, a Pierce 6X-histidine-tag antibody, an IgG-Cy3 antibody, and an IgG-Cy5 antibody were purchased from Thermo Fisher Scientific (Carlsbad, CA). Phospho-Tie2 antibodies (pTyr992 and pSer1119) were purchased from Cell Signaling Technology (Danvers, MA). [γ-³²P]-ATP was purchased from PerkinElmer (Waltham, MA).

Kennedy M.A., Xu Z., Wu Y, & Sohl C.D. (2019). A Tie2 kinase mutation causing venous malformations increases phosphorylation rates and enhances cooperativity. Biochemical and biophysical research communications, 509(4), 898-902.

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Isolation and Culture of Human Islets

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Human islets from normal donors were obtained from the University of Alberta Diabetes Institute, the University of Chicago Diabetes Research and Training Center, and the University of Minnesota Schulze Diabetes Institute. The age, sex, body mass index, and %HbA1c of each donor is provided in Table S1. Human islets were cultured in glutamine-free CMRL supplemented with 10 mM niacinamide and 16.7 μM zinc sulfate (Sigma), 1% ITS supplement (Corning), 5 mM sodium pyruvate, 1% Glutamax, 25 mM HEPES (American Bio), 10% HI FBS and antibiotics (10,000 units/ml penicillin and 10 mg/ml streptomycin). All media components were obtained from Invitrogen unless otherwise indicated.

Lewandowski S.L., Cardone R.L., Foster H.R., Ho T., Potapenko E., Poudel C., VanDeusen H.R., Sdao S.M., Alves T.C., Zhao X., Capozzi M.E., de Souza A.H., Jahan I., Thomas C.J., Nunemaker C.S., Davis D.B., Campbell J.E., Kibbey R.G, & Merrins M.J. (2020). Pyruvate kinase controls signal strength in the insulin secretory pathway. Cell metabolism, 32(5), 736-750.e5.

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Assessing Human Islet Responses to Inflammatory Stimuli

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Human islets were obtained from adult, nondiabetic organ donors from Prodo Laboratories, Inc., or the Integrated Islet Distribution Program at City of Hope. Islets were cultured in CMRL 1066 medium (Gibco) supplemented with 10% FBS (MilliporeSigma), 10 mM HEPES (AmericanBio), 2 mM l-glutamine (MilliporeSigma), and 1% pen-strep (Gibco). Where indicated, islets were treated with 25 or 100 ng/mL IFN-γ (R&D Systems) or 10 ng/mL TNF-α (R&D Systems). In some experiments, islets were pretreated with 5 μM ruxolitinib (Selleckchem) for 1 hour prior to the addition of IFN-γ. Islets were harvested at indicated time points and dissociated into single-cell suspensions using 0.05% trypsin-EDTA (Gibco). Cells were stained with FluoZin-3 (Invitrogen) and TMRE (Life Technologies) for β cell isolation experiments and sorted using a FACSAria II (BD).

Perdigoto A.L., Deng S., Du K.C., Kuchroo M., Burkhardt D.B., Tong A., Israel G., Robert M.E., Weisberg S.P., Kirkiles-Smith N., Stamatouli A.M., Kluger H.M., Quandt Z., Young A., Yang M.L., Mamula M.J., Pober J.S., Anderson M.S., Krishnaswamy S, & Herold K.C. (2022). Immune cells and their inflammatory mediators modify β cells and cause checkpoint inhibitor–induced diabetes. JCI Insight, 7(17), e156330.

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Isolating human pancreatic beta cells

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Human islets were obtained from adult, non-diabetic organ donors from Prodo Laboratories (Aliso Viejo, CA). Islets were cultured in CMRL1066 culture medium (Gibco) supplemented with 10% fetal bovine serum (Sigma), 10 mM Hepes (AmericanBio), 2mM L-glutamine (Sigma), and 1% Pen-Strep (Gibco). To modeling pancreas inflammation, human islets were treated with 25 ng/mL IFNγ (R&D) and/or 10 ng/mL TNFα (R&D) for 48 h and then dissociated into single cell suspensions using 0.05% trypsin-EDTA (Gibco). Cells were stained with FluoZin-3 (Invitrogen) and TMRE (Life Technologies) and sorted using a FACS Aria II (BD) and analyzed with FACSDiva (BD) and FlowJo (Tree Star Inc). In brief, pancreatic islet cells were first gated on SSC-A/FSC-H. Second, cells were gated on Zinc+ (ie FITC channel) cells. Third, cells were passed to a FSC-W/FSC-H and subsequently SSC-W/SSC-H to exclude doublets. Cells were then collected by zinc+ and TMRE+ cells to enrich viable beta cells.

Yang M.L., Horstman S., Gee R., Guyer P., Lam T.T., Kanyo J., Perdigoto A.L., Speake C., Greenbaum C.J., Callebaut A., Overbergh L., Kibbey R.G., Herold K.C., James E.A, & Mamula M.J. (2022). Citrullination of glucokinase is linked to autoimmune diabetes. Nature Communications, 13, 1870.

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Maintenance of Diverse Cell Lines

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HEK293 and HeLa cells were maintained in DMEM high glucose (GIBCO) supplemented with 10% fetal bovine serum (FBS; GIBCO), 100 U/ml penicillin, and 100 µg/ml streptomycin in 5% carbon dioxide at 37°C. Rat insulinoma INS1 832/13 cells and INS1 832/13 cells expressing SNAP tagged proinsulin or CatD-GFP and INS1 832/13 cells dually expressing SNAP-tagged proinsulin and LyzC-GFP were maintained in RPMI 1640 (cat no. 11879-020; GIBCO) containing 11 mM glucose and supplemented with 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin, 10 mmol/l HEPES (americanBIO), 2 mmol/l Glutamax (GIBCO), 1 mmol/l sodium pyruvate (GIBCO) and 50 µmol/l β-mercaptoethanol (americanBIO) in 5% carbon dioxide at 37°C.

Parchure A., Tian M., Stalder D., Boyer C.K., Bearrows S.C., Rohli K.E., Zhang J., Rivera-Molina F., Ramazanov B.R., Mahata S.K., Wang Y., Stephens S.B., Gershlick D.C, & von Blume J. (2022). Liquid–liquid phase separation facilitates the biogenesis of secretory storage granules. The Journal of Cell Biology, 221(12), e202206132.

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