Nicotinamide

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Nicotinamide is a form of vitamin B3 that serves as a precursor for the coenzyme nicotinamide adenine dinucleotide (NAD) in biological systems. NAD is essential for various metabolic processes within cells, including energy production and DNA repair.

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661 protocols using nicotinamide

Differentiation of WJ-MSCs into IPCs

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After two to four passages, 1 × 10⁶ WJ-MSCs were induced to differentiate into IPCs using two protocols. The first protocol (A) was carried out as described previously with slight modifications [9 (link)]; cells were preinduced for 48 hours with 10 mmol/L nicotinamide (Sigma-Aldrich, USA) and 1 mmol/L β-mercaptoethanol (Sigma-Aldrich, USA) in 10 % FBS LG-DMEM, and then reinduced for another 24 hours with 10 mmol/L nicotinamide and 1 mmol/L β-mercaptoethanol in serum-free high glucose (HG)-DMEM.
The second protocol (B) started exactly as protocol A; the cells were preinduced for 48 hours with 10 mmol/L nicotinamide and 1 mmol/L β-mercaptoethanol in 10 % FBS LG-DMEM, and then reinduced for another 24 hours with 10 mmol/L nicotinamide and 1 mmol/L β-mercaptoethanol in serum-free HG-DMEM. However, this was followed by further induction for 7 days by 10 nmol/L exendin-4 (Sigma-Aldrich, USA) in serum-free HG-DMEM supplemented with 10 mmol/L nicotinamide and 1 mmol/L β-mercaptoethanol. Noninduced control WJ-MSCs were fed with complete growth medium (10 % FBS LG-DMEM) and kept for the same time as the differentiation protocol following the same culturing conditions as described earlier in this study.

Kassem D.H., Kamal M.M., El-Kholy A.E, & El-Mesallamy H.O. (2016). Exendin-4 enhances the differentiation of Wharton’s jelly mesenchymal stem cells into insulin-producing cells through activation of various β-cell markers. Stem Cell Research & Therapy, 7, 108.

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Vitamin B Compounds Tested on C. teleta Larvae

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Thiamine HCl (B₁), riboflavin (B₂), nicotinamide (B₃), nicotinic acid (another form of B₃), pyridoxine HCl (B₆), biotin (B₇) and cobalamin (B₁₂) were obtained from Sigma Chemical Co. Thiamine HCl, nicotinic acid, pyridoxine HCl, biotin, and cobalamin were all tested on larvae of C. teleta at 500 µM. nicotinic acid was also tested at 1 mM. riboflavin was tested on larvae of C. teleta at 10, 50, 100, 150, and 200 µM. nicotinamide was tested on larvae of C. teleta at 2, 3, 4, 5, 6, 7, and 8 µM. The concentrations tested for nicotinamide and riboflavin were based on preliminary pilot studies. The photo-degradation product of riboflavin, lumichrome, was obtained from Sigma Chemical and tested at concentrations of 100, 500, and 1000 µM. The pyridine receptor antagonists and agonists 4-acetylpyridine, pyrazinecarboxamide, and β-NAD were all purchased from Sigma Chemical Co. and tested in final concentrations as stated in results. The serotonin receptor antagonist ketanserin was purchased from Sigma Chemical Co. and prepared as a 10 mM stock solution in ethanol.

Burns R.T., Pechenik J.A., Biggers W.J., Scavo G, & Lehman C. (2014). The B Vitamins Nicotinamide (B3) and Riboflavin (B2) Stimulate Metamorphosis in Larvae of the Deposit-Feeding Polychaete Capitella teleta: Implications for a Sensory Ligand-Gated Ion Channel. PLoS ONE, 9(11), e109535.

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Examining RORγt Acetylation by p300 in Transfected 293T Cells

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293T cells were transfected at 70–80% confluency with vectors for Flag-RORγt, HA-SIRT1, and myc-p300 (pcDNA3.1) in a 1:2:4 ratio by either calcium phosphate or Lipofectamine 2000 (Invitrogen), as indicated. Total transfected DNA for each sample was normalized by adding empty vector DNA. 6–8 h after transfection, cells were washed and media was replaced followed by treatment with TSA (400 nM) for 18 h to induce p300 hyperacetylation of RORγt. Cells were harvested in p300 lysis buffer (250 mM NaCl, 0.1% NP-40, 20 mM NaH₂PO₄, pH 7.5, 5 mM EDTA, 30 mM sodium pyrophosphate, 10 mM NaF, 5 mM nicotinamide, 400 nM TSA [Sigma-Aldrich], nicotinamide [5 mM; Sigma-Aldrich], and HALT protease/phosphatase inhibitors [Thermo Fisher Scientific]). After clarification, lysates were immunoprecipitated with α-Flag (M2) conjugated-agarose (Sigma-Aldrich), washed 5 times with lysis buffer and eluted with Flag peptide (100 µg/ml). Samples were boiled in Laemmli buffer for SDS-PAGE and Western blotting, and membranes were probed with antibodies against Flag (M2, Sigma-Aldrich) and pan-acetyllysine (9441; Cell Signaling Technology).

Lim H.W., Kang S.G., Ryu J.K., Schilling B., Fei M., Lee I.S., Kehasse A., Shirakawa K., Yokoyama M., Schnölzer M., Kasler H.G., Kwon H.S., Gibson B.W., Sato H., Akassoglou K., Xiao C., Littman D.R., Ott M, & Verdin E. (2015). SIRT1 deacetylates RORγt and enhances Th17 cell generation. The Journal of Experimental Medicine, 212(5), 607-617.

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Nicotinamide and Arsenic Exposure Protocol

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A 5mM nicotinamide (Sigma-Aldrich, St Louis, MO, USA) in double distilled water solution and a PBS (phosphate buffered solution) control were blinded and diluted 1 in 100 with cell media to a final concentration of 50μM. The cells and skin were incubated for 24h (±nicotinamide) prior to sodium arsenite exposure. A sodium arsenite (Ajax, Thermo Fisher Scientific Inc., Waltham, MA, USA) diluted in double distilled water solution and a PBS control were blinded and diluted in media to a final concentration of 2μM. 4 blinded culture environments resulted: nicotinamide + arsenic, nicotinamide alone, arsenic alone, control. The cells and tissue were then incubated in these media for another 24h prior to irradiation (or kept unirradiated) and replaced with the same culture environments post irradiation (Fig. 1).

Thompson B.C., Halliday G.M, & Damian D.L. (2015). Nicotinamide Enhances Repair of Arsenic and Ultraviolet Radiation-Induced DNA Damage in HaCaT Keratinocytes and Ex Vivo Human Skin. PLoS ONE, 10(2), e0117491.

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Circadian Rhythms in Leaf Development

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Analysis of circadian rhythms of the first true leaves was performed as described in 16 (link) without experimenter knowledge of seed lines. In those experiments where nicotinamide was applied, 50 mM nicotinamide (Sigma) or deionized water was applied once a day for 2 days before the start of imaging; 50 µl of solution was applied to the aerial parts of each seedling. The experiments were repeated twice but for the triple mutants, cml23-2 cml24-4 che-2 and cml23-2 cml24-4 toc1-2 were repeated three times. For cml23-2 cml24-4 toc1-2 two independent lines were used.

Ruiz M.C., Hubbard K.E., Gardner M.J., Jung H.J., Aubry S., Hotta C.T., Mohd-Noh N.I., Robertson F.C., Hearn T.J., Tsai Y.C., Dodd A.N., Hannah M., Carré I.A., Davies J.M., Braam J, & Webb A.A. (2018). Circadian oscillations of cytosolic free calcium regulate the Arabidopsis circadian clock. Nature plants, 4(9), 690-698.

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hESC-derived DE and HBs Expansion

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hESC-derived DE and HBs were treated with Accutase and re-seeded onto hydrogel substrates with varying stiffness, which were coated by 1:50 vitronectin for DE and 1:100 Matrigel for HBs. Cells were re-seeded at a density of 5 × 10⁴/cm² in medium supplemented with 10uM Y27632. Expansion medium for DE contained DMEM/F12 (Gibco) supplemented with 2% KSR, 50 ng/mL bone morphogenetic protein (BMP)-4 (Peprotech), 10 ng/mL epidermal growth factor (EGF; Peprotech), 10 ng/mL vascular endothelial growth factor (V EGF; Peprotech), 50 μg/mL L-ascorbic acid (Sigma-Aldrich), 50 μg/mL Vitamin A (TargetMol), 10 mM nicotinamide (Sigma-Aldrich), 1x GlutaMax and 1% penicillin-streptomycin. Expansion medium for HBs contained DMEM/F12 supplemented with 10% FBS, 1x insulin/transferrin/selenium (ITS, Sigma-Aldrich), 10 mM nicotinamide (Sigma-Aldrich), 10^− 7 M dexamethasone, 1x GlutaMax, 1% penicillin/streptomycin, 40 ng/mL hepatocyte growth factor (HGF; Peprotech) and 20 ng/mL EGF. Both DE and HB expansion media were changed daily.

Guo A., Wang B., Lyu C., Li W., Wu Y., Zhu L., Bi R., Huang C., Li J.J, & Du Y. (2020). Consistent apparent Young’s modulus of human embryonic stem cells and derived cell types stabilized by substrate stiffness regulation promotes lineage specificity maintenance. Cell Regeneration, 9, 15.

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Dental Stem Cells to Pancreatic Beta Cells

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The first protocol was carried out as described by Kassem et al. in 2016 [16 (link)]. Briefly, 2 × 10⁵ dental derived stem cells were pre-induced for 48 hours with 10 mmol/L nicotinamide (Sigma-Aldrich, USA) and 1 mmol/L β-mercaptoethanol (Sigma-Aldrich) in 10% FBS LG-DMEM, and then re-induced for another 24 hours with 10 mmol/L nicotinamide and 1 mmol/L β-mercaptoethanol in serum-free high glucose (HG)-DMEM (Serox). This was followed by further induction for 7 days by 10 nmol/L exendin-4 (Sigma-Aldrich) in serum free HG-DMEM supplemented with 10 mmol/L nicotinamide and 1 mmol/L β-mercaptoethanol (Fig. 1).Fig. 1

Schematic diagram outlining the differentiation protocol 1 for DPSCs & PDLSCs into pancreatic β cells. Factors required for each stage of differentiation are outlined

Aly R.M., Aglan H.A., Eldeen G.N, & Ahmed H.H. (2022). Optimization of differentiation protocols of dental tissues stem cells to pancreatic β-cells. BMC Molecular and Cell Biology, 23, 41.

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Directed Differentiation of Retinal Pigment Epithelium

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For directed differentiation of RPE, 10 mM nicotinamide (Sigma-Aldrich) /62 ng/ml activin A (Peprotech) based protocols were used according to previously described protocols^{49 (link)}. Confluent iPSCs cultures were switched to differentiation media containing: 78% Knockout DMEM (ThermoFisher, 10829018), 20% Knockout Serum Replacement (ThermoFisher, 10828028), 1% L-Glutamine (ThermoFisher, 25030081), 1% PenStrep (ThermoFisher, 15140-122), 800mg nicotinamide (Sigma, N0636). Differentiating iPSCs are maintained in differentiation media for 3 weeks, then switched to differentiation media plus 140ng/mL activin A (PeproTech, 120-14E) two weeks, then switched back to differentiation media. Following differentiation, pigmented colonies were manually excised and plated on growth factor reduced matrigel (corning) for expansion. RPE were passaged once at 1 to 6 for expansion before for being passaged and plated for assay conditions. For experiments on transwell inserts, we used 0.4uM pore sized coated with fibronectin (Corning 47743-654). RPE were plated in assay conditions at 50% confluency. In each assay RPE were plated at the same time and matured for a minimum of 12 weeks past the point of confluency before being assayed.

Eade K., Gantner M.L., Hostyk J.A., Nagasaki T., Giles S., Fallon R., Harkins-Perry S., Baldini M., Lim E.W., Scheppke L., Dorrell M.I., Cai C., Baugh E.H., Wolock C.J., Wallace M., Berlow R.B., Goldstein D.B., Metallo C.M., Friedlander M, & Allikmets R. (2021). Serine biosynthesis defect due to haploinsufficiency of PHGDH causes retinal disease. Nature metabolism, 3(3), 366-377.

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NMR Analysis of Aloe Vera Acetylation

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The degree of acetylation of the acemannan polymer, as the main component of AIR-samples, was analysed using the method described by Bozzi et al. (2007) [30 (link)]. Therefore, 2 mg of hydrolysed sample together with 2 mg nicotinamide (Sigma-Aldrich, St. Louis, MO, USA) were dissolved in heavy water (Sigma-Aldrich, St. Louis, MO, USA); nicotinamide was used as an internal shift standard, because its peaks appear after 7.5 ppm, which are well-separated from other Aloe vera derived proton peaks [31 (link)]. Then, the solubilized samples were transferred into Wildman Economic 5-mm NMR tubes. The ¹H-NMR spectra at 300.13 MHz were recorded on an Avance 300 spectrometer (Bruker, Billerica, MA, USA), equipped with a 5-mm broadband multinuclear z-gradient (BBO) probe head. The area under the curve (AUC) was calculated using the acetyl group signal corresponding to nicotinamide. The relative degree of acetylation of processed samples in relation to the fresh (reference) sample was calculated using the following Equation:
Representative ¹H-NMR-spectra from different Aloe vera samples to determine the degree of acetylation of acemannan can be found in the Figures S2 and S3 of the Supplementary Materials.

López Z., Salazar Zúñiga M.N., Femenia A., Acevedo-Hernández G.J., Godínez Flores J.A., Cano M.E, & Knauth P. (2022). Dry but Not Humid Thermal Processing of Aloe vera Gel Promotes Cytotoxicity on Human Intestinal Cells HT-29. Foods, 11(5), 745.

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Induction of Type 2 Diabetes in Rats

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Type 2 diabetes was infused in Wistar male rats that have fasted overnight, by injecting 60 mg/kg STZ (Sigma Aldrich, USA) 15 min after an injection of 110 mg/kg of nicotinamide (Sigma Aldrich, USA).[19 (link)] STZ and nicotinamide were dissolved in citrate buffer (pH 4.5) and saline.[21 (link)22 (link)] The ND control group was injected with citrate buffer.[20 (link)] After 1 week, blood was taken from the rats' tail veins for diagnosis of diabetes. Levels of fasting plasma glucose above 126 mg/dl were considered as evidence of type 2 diabetes.[23 (link)]

Dastbarhagh H., Kargarfard M., Abedi H., Bambaeichi E, & Nazarali P. (2019). Effects of food restriction and/or aerobic exercise on the GLUT4 in type 2 diabetic male rats. International Journal of Preventive Medicine, 10, 139.

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