Krebs buffer was composed of (in mM): 146.9 NaCl, 4.7 KCl, 2 CaCl2, 1.2 MgSO4, 1.2 NaH2PO4•H2O, 3 NaHCO3, 1.5 NaHEPES, and 5 d-glucose (pH = 7.4 at 37°C). Krebs-BSA buffer was prepared with the addition of 0.5% (w/v) bovine serum albumin (BSA) while Krebs Ca2+-free replaced CaCl2 with 3mM EGTA. Tamoxifen was dissolved to 10mg/ml in a Safflower Oil-Ethanol (95%-5% v/v) solution with rocking agitation, separated into aliquots, and stored at −20 °C. Wortmannin was dissolved in DMSO to a stock solution of 1 mM. Pinacidil was dissolved in DMSO to a stock concentration of 1 μM. Nifedipine was dissolved in DMSO to a stock concentration of 1 mM. All chemicals were obtained from Sigma (St. Louis, MO), with the exception of BSA (US Biochemicals; Cleveland, OH), MgSO4 and NaHEPES (Fisher Scientific; Pittsburgh, PA).
Na hepes
Manufactured by Thermo Fisher Scientific
Sourced in United States
Na-HEPES is a commonly used buffer solution for maintaining pH in various biological and biochemical applications. It is a sodium salt of the N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer. Na-HEPES is designed to maintain a stable pH around 7.5 in aqueous solutions.
Automatically generated - may contain errors
Lab products found in correlation
Mgso4, by Thermo Fisher Scientific (11 mentions)
Ca no3 2, by Merck Group (1 mentions)
Nano3, by Merck Group (1 mentions)
Raw264, by American Type Culture Collection (1 mentions)
Sodium pyruvate, by Lonza (1 mentions)
Iberiotoxin, by Bio-Techne (1 mentions)
Apamin, by Alomone (1 mentions)
Ns11021, by Bio-Techne (1 mentions)
Bekm 1, by Alomone (1 mentions)
E 4031, by Bio-Techne (1 mentions)
Glibenclamide glib, by Merck Group (1 mentions)
Pinacidil, by Merck Group (1 mentions)
Glibenclamide, by Merck Group (1 mentions)
14 protocols using na hepes
1
Krebs Buffer and Compound Preparation
2
Preparation of Krebs Buffers and Compounds
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Krebs buffer was composed of (in mM): 146.9 NaCl, 4.7 KCl, 2 CaCl2, 1.2 MgSO4, 1.2 NaH2PO4•H2O, 3 NaHCO3, 1.5 NaHEPES, and 5 d-glucose (pH = 7.4 at 37°C). Krebs-BSA buffer was prepared with the addition of 0.5% (w/v) bovine serum albumin (BSA) while Krebs Ca2+-free replaced CaCl2 with 3mM EGTA. Tamoxifen was dissolved to 10mg/ml in a Safflower Oil-Ethanol (95%–5% v/v) solution with rocking agitation, separated into aliquots, and stored at −20 °C. Wortmannin was dissolved in DMSO to a stock solution of 1 mM. Pinacidil was dissolved in DMSO to a stock concentration of 1 μM. Nifedipine was dissolved in DMSO to a stock concentration of 1 mM. All chemicals were obtained from Sigma (St. Louis, MO), with the exception of BSA (US Biochemicals; Cleveland, OH), MgSO4 and NaHEPES (Fisher Scientific; Pittsburgh, PA).
3
Cerium Biouptake and Speciation Protocol
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Experimental media. All Ce solutions were prepared from a 1000 mg L -1 stock solution of Ce(NO3)3 in 7% HNO3 (Inorganic Ventures). Stock solutions with lower concentrations were kept in 2% ultrapure nitric acid (BDH) in Milli-Q water (R>18 M cm; dissolved organic carbon<2 g C L -1 ). Biouptake and speciation measurements were performed in pH controlled media composed of 0.01 M NaNO3 (Sigma) for pH 4.0, 0.01 M NaMES (2-(Nmorpholino)ethanesulfonic sodium salt, Acros Organics) for pH 5 and 6 and 0.01 M NaHEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic sodium salt, Fisher bioreagents) for pH 7 and 8. All media contained 1x10 -5 M of Ca(NO3)2 (Sigma Aldrich) in order to help preserve the integrity of the biological cell wall [22] . Following their preparation, solutions were placed on a rotary shaker (100 rpm) for 24h. For experiments involving natural organic matter, a Suwannee River fulvic acid standard (SRFA, 2S101F) was used. Stock solutions of 250 mg L -1 of SRFA were prepared by dissolving the lyophilized solid in the experimental media described above at pH 5.0, 6.0 or 7.0. Following a 24 hour equilibration, stock solutions were then diluted into buffered experimental media for use after another 24 h. All polymerware was soaked a minimum of 24 hours in 2% HNO3 then rinsed 7x with MilliQ grade water, before drying under laminar flow conditions.
4
Mycobacterial Growth and Macrophage Infection
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M. bovis BCG and M. tuberculosis H37Rv strains were grown in Middlebrook 7H9 broth medium (Difco, Becton-Dickinson) containing 10% OADC (oleic acid/albumin/dextrose/catalase, 0.05% Tween 80) until exponential phase and then aliquoted and stored at −70 °C until use. Prior to use, the bacterial stocks were thawed and the mycobacteria were de-clumped by a brief sonication and passed through a syringe fitted with a 27-gauge needle at least 10 times. The macrophage cell line RAW 264.7 (ATCC, Manassas, VA) was maintained in Dulbecco modified Eagle’s minimal essential medium (DMEM, Cellgro, Manassas, VA) supplemented with 10% fetal bovine serum (Hyclone, South Logan, Utah), 25 mM Na-HEPES (ThermoScientific, Rockford, IL), 1 mM Sodium pyruvate (Lonza, Walkersville, MD), 100 U/mL penicillin and 100 U/mL streptomycin (Hyclone, South Logan, Utah) at 37 °C with 5% CO2. Bone marrow-derived macrophages (BMMs) were prepared from wild type C57BL/6 or Rab27a deficiency mice as described previously29 (link).
5
Krebs Buffer Vasodilation Protocol
6
Krebs Buffer Composition and Application
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Krebs buffer contained: 146.9 mM NaCl, 4.7 mM KCl, 2 mM CaCl2·2H2O, 1.2 mM MgSO4, 1.2 mM NaH2PO4·H2O, 3 mM NaHCO3, 1.5 mM Na-HEPES, and 5 mM D-glucose (pH = 7.4). An identical buffer was prepared with the addition of 0.5% bovine serum albumin (“Krebs-BSA”). During cannulation Krebs-BSA was present both luminally and abluminally; during the experiment the abluminal solution was constantly exchanged with Krebs buffer. Ca2+-free Krebs was Krebs with 3 mM EGTA replacing CaCl2·2H2O and used to determine passive diameter. All chemicals were obtained from Sigma-Aldrich (St. Louis, MO), with the exception of BSA (US Biochemicals; Cleveland, OH), MgSO4 and Na-HEPES (ThermoFisher Scientific; Pittsburgh, PA).
7
Tissue Perfusion Buffer Preparation
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Krebs buffer was composed of (in mM) 146.9 NaCl, 4.7 KCl, 2 CaCl2•2H2O, 1.2 MgSO4, 1.2 NaH2PO4•H2O, 3 NaHCO3, 1.5 NaHEPES, and 5 d-glucose, pH 7.4, at 37°C. Krebs-BSA buffer was prepared with the addition of 0.5% (wt/vol) BSA. During cannulation the luminal and bath solutions contained Krebs solution with BSA, but during the experiment, the bath solution was constantly exchanged with Krebs solution without albumin. For the Ca2+-free Krebs solution, 3 mM EGTA replaced CaCl2•2H2O. A benzbromarone stock solution was prepared at 10 mM in DMSO, separated into aliquots, and stored at −20°C for up to 2 mo. Doxycycline was prepared at 4 mg/ml in sterile saline and separated into aliquots, and frozen at −20°C until use. Tamoxifen was dissolved to 10 mg/ml in a safflower oil–ethanol (95/5% vol/vol) solution with rocking agitation, separated into aliquots, and stored at −20°C. Wortmannin was dissolved in DMSO to a stock solution of 10 mM. All chemicals were obtained from Sigma-Aldrich, with the exception of BSA (United States Biochemicals), and MgSO4 and NaHEPES (Thermo Fisher Scientific).
8
Characterization of Cardiac Ion Channels
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Krebs buffer contained: 146.9 mM NaCl, 4.7 mM KCl, 2 mM CaCl2·2H2O, 1.2 mM MgSO4, 1.2 mM NaH2PO4·H2O, 3 mM NaHCO3, 1.5 mM Na-HEPES, and 5 mM D-glucose (pH 7.4). An identical buffer (“Krebs-BSA”) also contained 0.5% bovine serum albumin. Krebs-BSA buffer was present both luminally and abluminally during cannulation, but the abluminal solution was constantly exchanged with plain Krebs during the experimental protocol. For Ca2+-free Krebs, 3 mM EGTA replaced CaCl2·2H2O. All chemicals were obtained from Sigma-Aldrich (St. Louis, MO) except BSA (US Biochemicals; Cleveland, OH), MgSO4 and Na-HEPES (ThermoFisher Scientific). E-4031 was obtained from Tocris (#1808) and dissolved in water to make a 1 mM stock solution. BeKm-1 was obtained from Alomone Labs (#STB-470) and dissolved in water to make a 100 mM stock solution. RPR-260243, type 1 ERG channel agonist, was obtained from MedChemExpress (#HY-16915) and dissolved in DMSO to make a 10 mM stock solution. ICA-105574, a type 2 ERG channel agonist, was obtained from Abious (#AOB2681) and dissolved in DMSO to make a 10 mM stock solution.
9
Krebs-BSA Buffer for Vascular Perfusion
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Krebs buffer contained: 146.9 mM NaCl, 4.7 mM KCl, 2 mM CaCl2·2H2O, 1.2 mM MgSO4, 1.2 mM NaH2PO4·H2O, 3 mM NaHCO3, 1.5 mM Na-HEPES, and 5 mM D-glucose (pH = 7.4). An identical buffer was prepared with the addition of 0.5% bovine serum albumin (BSA). During cannulation, Krebs-BSA buffer was present both luminally and abluminally; however, during the experiment, the abluminal solution was constantly exchanged with plain Krebs buffer. All chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA), with the exception of BSA (US Biochemicals; Cleveland, OH, USA), MgSO4, and Na-HEPES (ThermoFisher Scientific; Pittsburgh, PA, USA).
10
Calcium-Dependent Krebs Buffer Protocol
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Krebs buffer contained: 146.9 mM NaCl, 4.7 mM KCl, 2 mM CaCl2·2H2O, 1.2 mM MgSO4, 1.2 mM NaH2PO4·H2O, 3 mM NaHCO3, 1.5 mM Na-HEPES, and 5 mM d -glucose (pH = 7.4). An identical buffer (“Krebs-BSA”) also contained .5% bovine serum albumin. Krebs-BSA buffer was present both luminally and abluminally during cannulation, but the abluminal solution was constantly exchanged with plain Krebs during the experimental protocol. For Ca2+-free Krebs, 3 mM EGTA replaced CaCl2·2H2O. All chemicals were obtained from Sigma-Aldrich (St. Louis, MO) except BSA (US Biochemicals; Cleveland, OH), MgSO4 and Na-HEPES (ThermoFisher Scientific; Pittsburgh, PA).
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