Resting and activated mouse neutrophils (4 × 106 per ml) were treated with and without glycolysis (2-DG) or CI (rotenone) inhibitors for 70 min at 37oC in DMEM medium (no glucose, no glutamine, no phenol red (Cat # A14430-01, Thermo Fisher Scientific). 12 mM D-glucose was added for lactate and ATP/ADP measurements. For NAD+/NADH quantitative analysis, cells were treated as described above, but cultured in PBS. Upon activation, cells were placed on ice for lactate analysis or the reaction was stopped by adding Krebs-Ringer bicarbonate buffer (Cat # K4002, Sigma-Aldrich) and cell pellets were resuspended in Krebs-Ringer bicarbonate buffer with 1% TCA (for ATP/ADP) or cold PBS (for NAD+/NADH). The ATP/ADP ratio was measured by Cellular ADP/ATP‐Glo™ Assay (Promega AG), according to the instructions of the manufacturer. Lactate and the NAD+/NADH ratio were measured for luciferase activity by Lactate-Glo™ Assay or NAD/NADH‐Glo™ Assay (Promega AG), according to the instructions of the manufacturer. Values were normalized to the total protein content.
Krebs ringer bicarbonate buffer
Manufactured by Merck Group
Sourced in United States, United Kingdom
Krebs-Ringer bicarbonate buffer is a physiological salt solution that is commonly used in cell and tissue culture applications. It contains a balanced mixture of salts, glucose, and bicarbonate to maintain the pH and osmotic environment necessary for the survival and normal function of cells and tissues.
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Lab products found in correlation
Rpmi 1640 medium, by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (3 mentions)
Ussing chamber, by Physiologic Instruments (2 mentions)
Sodium phosphate dibasic, by Thermo Fisher Scientific (2 mentions)
Sodium chloride (nacl), by Thermo Fisher Scientific (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
3 isobutyl 1 methyl xanthine (ibmx), by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Ultrasensitive insulin elisa kit, by Crystal Chem (1 mentions)
Trimethylamine, by Thermo Fisher Scientific (1 mentions)
Calcium chloride dehydrate, by Thermo Fisher Scientific (1 mentions)
Magnesium chloride hexahydrate, by Thermo Fisher Scientific (1 mentions)
Sodium phosphate monobasic monohydrate, by Thermo Fisher Scientific (1 mentions)
Sodium acetate, by Thermo Fisher Scientific (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (1 mentions)
Sodium alginate, by Merck Group (1 mentions)
Tug891, by Bio-Techne (1 mentions)
Ah7614, by Bio-Techne (1 mentions)
28 protocols using krebs ringer bicarbonate buffer
1
Glycolysis and Complex I Inhibition in Activated Mouse Neutrophils
2
Ussing Chamber Intestinal Permeability
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Mice were sacrificed at designated time points and proximal segments of the duodenum, jejunum, and ileum and distal colon were removed. Sections were mounted in Lucite chambers and placed in Ussing chambers (Physiologic Instruments, San Diego, CA) exposing mucosal and serosal surfaces to oxygenated (95% O2, 5% CO2) Krebs bicarbonate ringer buffer (Sigma, St. Louis, MO). Intestinal sections were not stripped of underlying muscle. Buffer was maintained at 37°C by a heated water jacket and samples were allowed to equilibrate for 30 min. Transepithelial conductance (Gt) was measured by clamping the voltage and recording the change in the short‐circuit current (Isc) following a pulsed command voltage every 20 sec.
For measurements of tissue flux, 4 kDa FITC‐dextran (2.2 mg/mL final concentration) was added to the mucosal chamber; 10 kDa rhodamine B isothiocyanate (RITC)‐dextran (0.55 mg/mL final concentration) was also added to the mucosal chamber and used as a control for tissue integrity. Serosal chamber samples were taken at 0 and 60 min, and fluorescence intensity determined (FITC excitation, 485 nm; emission, 530 nm; RITC excitation, 595 nm; emission, 615 nm; Tecan). FITC‐dextran/RITC‐dextran concentrations were determined using a standard curve and FITC‐dextran flux in OVX mice normalized against sham mice and reported as fold change.
For measurements of tissue flux, 4 kDa FITC‐dextran (2.2 mg/mL final concentration) was added to the mucosal chamber; 10 kDa rhodamine B isothiocyanate (RITC)‐dextran (0.55 mg/mL final concentration) was also added to the mucosal chamber and used as a control for tissue integrity. Serosal chamber samples were taken at 0 and 60 min, and fluorescence intensity determined (FITC excitation, 485 nm; emission, 530 nm; RITC excitation, 595 nm; emission, 615 nm; Tecan). FITC‐dextran/RITC‐dextran concentrations were determined using a standard curve and FITC‐dextran flux in OVX mice normalized against sham mice and reported as fold change.
3
Colon Tissue Permeability Assessment
4
Glucose-Stimulated Insulin Secretion Assay
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The islets were transferred to a 96-well plate, and the culture media was replaced with Krebs-Ringer bicarbonate buffer (Sigma Aldrich, Taufkirchen, Germany, cat. no. K4002) modified with HEPES (KRBH) containing 129 mM NaCl, 4.8 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4·H2O, 2 mM CaCl2, 24 mMNaHCO3, 6 mM HEPES, and 0.2% bovine serum albumin with PH adjusted to 7.4. Then, the islets were incubated for 1 h in starving glucose media (KRBH with 1 mM Glucose) before starting the GSIS. Different glucose concentrations (2.8 and 16.8 mM) were added to the islets (for 1 h each). For compound treatment, a concentration of 10 nM of GLP-1 or GIP was added during the GSIS. The supernatants were collected and used for insulin measurement. Islets were lysed for DNA measurements. Insulin levels were measured with the ultrasensitive insulin ELISA kit (Crystal Chem, Zaandam, Netherlands, cat. no. 90080). The data was normalized to the DNA content.
5
Development of Nicotine-Loaded Buccal Strips
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Hydroxypropylmethylcellulose (HPMC; Methocel K100 Premium LV) was a gift from Colorcon Limited (Dartford, UK), and magnesium aluminium silicate (MAS) was a gift from R.T. Vanderbilt Company Inc. (Norwalk, CT, USA). Sodium hydroxide, potassium dihydrogen phosphate, and gelatine were purchased from Fluka Analytical (Buchs, Switzerland). Nicotine (liquid form), sodium alginate (SA; molecular weight 120,000–190,000 g/mol, mannuronate/guluronate ratio 1.56), submaxillary mucin from porcine stomach, PBS tablets (pH 7.4), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Krebs–Ringer bicarbonate buffer, and dimethyl sulfoxide (DMSO) were all obtained from Sigma Aldrich (Dorset, UK). Sodium acetate, trimethylamine, and glycerol (GLY) were purchased from Fisher Scientific (Loughborough, UK). Commercially available NIC-loaded strips (NiQuitin®) was purchased from a local pharmacy (Gillingham, Kent). Calcium chloride dehydrate, sodium chloride, sodium phosphate dibasic, magnesium chloride hexahydrate, potassium carbonate hemihydrate, sodium phosphate monobasic monohydrate, Sodium acetate, and trimethylamine were all purchased from Fisher Scientific (Loughborough, UK). The EpiOralTM buccal tissue kit (ORL-200) was purchased from MatTek Corporation (Ashland, MA, USA).
6
Calcium Signaling in Differentiated Cells
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Differentiated cells were incubated for 1 h at RT with the calcium‐sensitive dye Fluo‐4‐AM (Invitrogen) in Krebs–Ringer bicarbonate buffer (Sigma). Hereafter, the cells were washed twice with buffer, followed by live cell imaging with a confocal laser scanning microscope (LSM 510, Zeiss) and stimulation with TUG‐891 (10 μM), with or without preincubation with the GPR120 antagonist AH7614 for 5 min (100 μM; Tocris) or the Gαq inhibitor YM‐254890 for 30 min (0.1 μM, Alpha Laboratories).
7
Neonatal Mouse Testis Explant Culture
8
Cyclic AMP Assay in GLP-1 and GIP Cell Lines
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Example 2
Cyclic AMP Assay
The following materials were used: cAMP Hunter™ CHO-K1 GLP-1 or GIP cell lines (DiscoveRx Corporation), Ham's F-12 (Gibco Cat. #21765), 10% heat inactivated FBS (Gibco Cat #16000), Penicillin/Streptomycin/L-Glutamine (Gibco Cat #10378) and 800 μg/ml G418 (geneticin, Gibco Cat. #10131).
CHO-K1 cells expressing GLP-1 or GIP receptors were suspended in 10 ml assay buffer (Krebs-Ringer bicarbonate buffer (Sigma-Aldrich Cat. # K4002) containing 0.5 mM IBMX (Sigma-Aldrich Cat#17018) and 0.1% BSA (Sigma-Aldrich Cat. # A-2153)) at a cell density of 100,000 cells/ml. The cell suspension (25 μl) was subsequently transferred to a half-area plate (Costar Cat. #3694) and drug solutions (25 μl) were added to the wells at appropriate concentrations. The cells were incubated for 30 min at room temperature on a plate shaker. The cAMP content was determined using the Cisbio “cAMP dynamic kit” following the manufacturer's instructions (Cisbio Bioassays, France). All experiments were performed in duplicates and drugs were tested at least twice (N≥2).
9
Oligomeric DOCA-Conjugated GLP-1A Synthesis
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Exenatide and GLP-1A-Cys were purchased from Anygen (Gwangju, Republic of Korea). Oligomeric deoxycholic acids (DOCAs) were synthesized from Mediplex (Seoul, Republic of Korea) with 4-methylmorpholine (4-MMP) (Sigma-Aldrich, St. Louis, MO, USA), dicyclohexylcarbodiimide (DCC) (Sigma-Aldrich), N-hydroxysuccinimide (NHS) (Sigma-Aldrich), lys(BOC)OMe and BOC-lysBOC-Osu, DOCA ethylenediamine [molecular weight (MW) of 434.7], bisDOCA ethylenediamine (MW of 937.4), and tetraDOCA ethylenediamine (MW of 1,942.9). For chemical conjugation of GLP-1A with oligomeric DOCAs, 6-maleimidohexanoic NHS, N,N-dimethylformamide (DMF), and triethylamine were purchased from Sigma-Aldrich. For in vitro experiments, Dulbecco’s modified Eagle’s medium (DMEM), phosphate-buffered saline (PBS), and Hank’s balanced salt solution (HBSS) were purchased from Corning Inc. (Corning, NY, USA). Krebs–Ringer bicarbonate buffer (KRBB) was purchased from Sigma-Aldrich. In addition, rabbit monoclonal anti-E cadherin (Abcam, Cambridge, UK), rabbit monoclonal anti-Rps20 (Abcam), anti-SLC10A2 rabbit polyclonal antibody (Bioss Antibodies, Woburn, MA, USA), and anti-rabbit immunoglobulin G–horseradish peroxidase secondary antibody (Sigma-Aldrich) were purchased for western blotting.
10
Primary Hepatocyte Isolation from Mice
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Age-matched C57BL/6J albino males between 3 and 4 months were used for PH culture isolation. All mice were anesthetized using a ketamine/xylazine mixture (Henry Schein) before isolation. The hepatic portal vein was cannulated and perfused with Kreb’s Ringer Bicarbonate Buffer (Sigma-Aldrich) containing EGTA for 10 min at 37 °C. After the first wash, a second Kreb’s Ringer wash containing CaCl2 and Liberase (Roche Holding AG) was applied for 10 min at 37 °C. Hepatocytes were filtered through a gauze mesh and resuspended in plating media: William’s Media E (Sigma-Aldrich) containing 10% fetal bovine serum (Sigma-Aldrich), 200 nM dexamethasone (Sigma-Aldrich), 1 × penicillin/streptomycin (Thermo Fisher Scientific), and 2 mM L-glutamine (Thermo Fisher Scientific). Cells were plated at a density of 3 × 105/ml on a six-well collagen (Sigma-Aldrich)-coated plate. Hepatocytes were allowed to recover overnight and all experiments were started 24 h post isolation, unless otherwise indicated.
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