Islets were isolated using collagenase P (Roche Applied Science) injected to the pancreatic duct, followed by Histopaque (1119 and 1077; Sigma-Aldrich) gradient. Islets were incubated overnight in standard RPMI-1640 medium (Biological Industries) supplemented with 10% FBS, L-glutamine, and penicillin-streptomycin in a 37°C, 5% CO2 incubator. Hand-picked islets (30–70 islets) were placed for 42–48 h in 5 mmol/L or 25 mmol/L glucose RPMI medium and treated with 325 μmol/L diazoxide (Sigma-Aldrich), 10 μmol/L nifedipine (Alomone Labs), 37 nmol/L tacrolimus (Astellas Pharma), 0.5 μmol/L glyburide (Sigma-Aldrich), or 3 μmol/L Bay-K8644 (Alomone Labs).
Nifedipine
Manufactured by Alomone
Sourced in United States
Nifedipine is a calcium channel blocker used as a reference standard in laboratory research. It inhibits the movement of calcium ions across cell membranes, which can affect various physiological processes.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640 medium, by Sartorius (1 mentions)
Histopaque, by Merck Group (1 mentions)
Glyburide, by Merck Group (1 mentions)
Diazoxide, by Merck Group (1 mentions)
Collagenase p, by Roche (1 mentions)
Bay k8644, by Alomone (1 mentions)
Tacrolimus, by Astellas Pharma (1 mentions)
Bapta am, by Merck Group (1 mentions)
U73122, by Merck Group (1 mentions)
Apamin, by Alomone (1 mentions)
Cyclopiazonic acid cpa, by Alomone (1 mentions)
Heparin, by MP Biomedicals (1 mentions)
ω conotoxin, by Alomone (1 mentions)
ω agatoxin, by Alomone (1 mentions)
Paxilline, by Alomone (1 mentions)
Hank s balanced salt solution, by Thermo Fisher Scientific (1 mentions)
Nickel 2 chloride, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Trypsin, by Merck Group (1 mentions)
4 protocols using nifedipine
1
Islet Isolation and Functional Assays
2
Electrophysiological Experiments: Chemical Modulations
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Depending on the purpose of the electrophysiological experiments, the chemicals applied via the recording solution included nifedipine (10 μM), ω-conotoxin (1 μM), ω-agatoxin (50 nM), apamin (100 nM), paxilline (10 μM), cyclopiazonic acid (CPA; 30 μM; all purchased from Alomone Labs, Jerusalem, Israel), BAPTA-AM (1 0 μM; Sigma), or U73122 (4 μM; Sigma). The pipette solution also included heparin (low molecular weight; 4 mg/ml; MP Biomedicals), ruthenium red, D-IP3, L-IP3 (100 μM; Alomone Labs), glutathione S-transferase (GST)-CaBP1 (16 nM; Abnova Corp.), calmodulin (3 μM; BioVision Inc.), CaBP1 antibody (10 μg/ml; Novus Biologicals), and calmodulin antibody (10 μg/ml; Novus Biologicals).
3
Isolation and Characterization of Cells
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Reagents were obtained from the following sources: collagenase P, bovine serum albumin (BSA), Ficoll 400, trypan blue, 4-(2-hydroxyethyl)-1 piperazineethanesulfonic acid (HEPES), poly-L-lysine, Staphylococcus aureus protein A, trypsin, insulin, guinea-pig complement, nickel chloride (II), and all salts of electrophysiological recordings from Sigma-Aldrich (St. Louis, MO, USA); mibefradil, nifedipine, anti-VGCC rabbit polyclonal IgG from Alomone Labs (Jerusalem, Israel); FITC-conjugated anti-guinea pig, and Alexa Fluor 647-conjugated anti-rabbit IgG from Jackson ImmunoResearch (West Grove, USA). Tetrodotoxin from Calbiochem (La Jolla, CA, USA); TTA-A2 kindly donated by Merck (West Point, PA, USA) to Dr. Juan Carlos Gomora; tissue culture dishes from Corning (Corning, NY, USA); Hanks’ balanced salt solution, RPMI-1640 salts, and penicillin–streptomycin solution, fetal bovine serum, and L-glutamine solution from Thermo Fisher Scientific Inc. (Massachusetts, USA).
4
Cytokine Quantification in Cell Cultures
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CCL-2 and IL-6 concentrations in cell culture supernatants were quantified by ELISA (BD Biosciences, San Jose, CA, USA) after the treatment of cells with 5 ng/mL or 25 ng/mL TNF-α (R&D Systems, Boston, MA, USA; E. coli-derived), NS1619 (30 μM; Millipore Sigma, Carlsbad, CA, USA), Nifedipine (10 μM; Alomone Labs, Jerusalem, Israel), JNK inhibitor VIII (20 μM; Millipore Sigma, Carlsbad, CA, USA), SB203580 (20 μM; Millipore Sigma, Carlsbad, CA, USA) or equimolar vehicle controls for 24 hrs. To avoid any potential cell toxicity related to TNF-α, in a pilot dose–response experiments, we treated HULEC5a cells with 5 and 50 ng/mL TNF-α for 24 hrs, measured CCL-2 and IL-6 concentrations in cell supernatants, and assessed cell viability (XTT Cell Viability Assay Kit, Biotium, Freemont, CA, USA).
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