Dapagliflozin

Manufactured by Cayman Chemical

Sourced in United States

Dapagliflozin is a sodium-glucose cotransporter 2 (SGLT2) inhibitor. It is used to measure the inhibitory activity of SGLT2, a key regulator of glucose homeostasis.

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Lab products found in correlation

Empagliflozin, by Adipogen (1 mentions) Anti atf4, by Cell Signaling Technology (1 mentions) Mouse monoclonal antibodies, by Cell Signaling Technology (1 mentions) C2 ceramide, by Cayman Chemical (1 mentions) Humulin n insulin, by Eli Lilly (1 mentions) Streptozotocin (stz), by Merck Group (1 mentions) Tgf β1, by Merck Group (1 mentions) U0126, by Merck Group (1 mentions) Glucose uptake cell based assay kit, by Cayman Chemical (1 mentions) Empagliflozin, by Cayman Chemical (1 mentions) Phloretin, by Merck Group (1 mentions) Sotagliflozin, by Cayman Chemical (1 mentions) Phlorizin, by Merck Group (1 mentions) Canagliflozin, by Mitsubishi (1 mentions) Ta 1887, by Mitsubishi (1 mentions) Cy3 conjugated anti rabbit secondary antibody, by Jackson ImmunoResearch (1 mentions) Penicillin streptomycin, by Sartorius (1 mentions) Rapamycin, by Cell Signaling Technology (1 mentions) A1r confocal microscope, by Nikon (1 mentions) Torin 1, by Cell Signaling Technology (1 mentions)

9 protocols using dapagliflozin

Diabetic PTC Model: Evaluating SGLT2 Inhibitors

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PTCs were proliferated to confluence in a standard cell culture medium (as described in Section 4.1). PTCs were then washed, placed in serum-free Medium 199 for 2 h, and stimulated as indicated in the respective assay. Stimulations were performed in high-glucose Medium 199 (HG, glucose content 450 mg/dL [25 mM]) with 10% FBS, which induced a diabetic milieu in the cultures.
For protein measurements in the supernatants (EIA, Section 4.6), stimulations were performed in HG without serum. For stimulations, empagliflozin (Adipogen, San Diego, CA, USA; No. AG-CR1-3619) and dapagliflozin (Cayman Chemical, Ann Arbor, MI, USA; No. 11574) were dissolved in dimethylsulfoxide (stock solution 1 mM) and diluted in the medium to 500 nM. This concentration of both gliflozins (500 nM) matched the published maximal therapeutic concentration observed (C_max) [51 ,52 ]. An inflammatory microenvironment was induced by culturing PTCs in HG with a mixture of cytokines (CM) containing γ-interferon (200 U/mL), interleukin-1β (25 U/mL), and TNF-α (10 ng/mL). Stimulations were performed for 24 or 48 h, as indicated.

Koch B., Fuhrmann D.C., Schubert R., Geiger H., Speer T, & Baer P.C. (2023). Gliflozins Have an Anti-Inflammatory Effect on Renal Proximal Tubular Epithelial Cells in a Diabetic and Inflammatory Microenvironment In Vitro. International Journal of Molecular Sciences, 24(3), 1811.

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Investigating ER Stress Signaling Pathways

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Dapagliflozin and C2 ceramide were purchased from Cayman Chemical (Ann Arbor, MI, USA) and SIGMA-ALDRICH (St. Louis, MO, USA) respectively. Rabbit polyclonal anti-elf2α, anti-phospho-elf2α Ser51, and anti-ATF4, and mouse monoclonal antibodies were from Cell Signaling Technology Inc. (Boston, MA, USA). Rabbit polyclonal anti-IRE1α and anti-phospho-IRE1α, and mouse monoclonal anti-ATF6 antibodies were from Novus Biologicals (Littleton, CO, USA). Mouse monoclonal anti-GAPDH antibody was from MBL (Woods Hole, MA, USA). All other reagents were purchased from Sigma-Aldrich.

Shibusawa R., Yamada E., Okada S., Nakajima Y., Bastie C.C., Maeshima A., Kaira K, & Yamada M. (2019). Dapagliflozin rescues endoplasmic reticulum stress-mediated cell death. Scientific Reports, 9, 9887.

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Diabetic Medication Administration Protocol

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Streptozotocin was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Insulin
(humulin N) was purchased from Eli Lilly & Co. (Tokyo, Japan). For administration,
insulin was diluted with physiological saline and administered subcutaneously.
Dapagliflozin was purchased from Cayman Chemical Co. (Ann Arbor, MI, USA). To prepare the
stock solution, Dapagliflozin was dissolved in ethanol, and then stored at −20°C. Prior to
use, Dapagliflozin was diluted in phosphate buffer saline and administered orally.

SAYED N., ABDALLA O., KILANY O., DESSOUKI A., YOSHIDA T., SASAKI K, & SHIMODA M. (2020). Effect of dapagliflozin alone and in combination with insulin in a rat model of type 1 diabetes. The Journal of Veterinary Medical Science, 82(4), 467-474.

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Glucose-Induced Stress and TGF-β1 Signaling

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Cells were serum starved for 24 h prior to treatment. All subsequent conditions were supplemented with 0.1% BSA. Cells were exposed to three different conditions: (i) 7 mM D-glucose (control); (ii) 7 mM D-glucose + 18 mM D-glucose (high D-glucose or D-Glu); and (iii) 7 mM D-glucose + 18 mM L- glucose (osmotic control or L-Glu), with or without TGF-β1 0.75 ng/ml (Sigma-Aldrich). We used L-glucose to account for the osmotic stress the cells are subjected to upon >7 mM glucose treatment as osmotic stress is known to activate ERK in renal epithelial cells [22 (link)]. The SGLT2 inhibitor dapagliflozin (Cayman Chemical) was administered at 0.1, 1, or 10 nM while MEK/ERK inhibitor U0126 (Sigma-Aldrich) was administered at 10 μM. Treatment times ranged from 5 min to 24 h.

Pan X., Phanish M.K., Baines D.L, & Dockrell M.E. (2021). High glucose–induced Smad3 linker phosphorylation and CCN2 expression are inhibited by dapagliflozin in a diabetic tubule epithelial cell model. Bioscience Reports, 41(6), BSR20203947.

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Glucose Uptake Assay in Hepatocytes

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Glucose uptake was assessed using a glucose uptake cell-based assay kit (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer’s instructions. Huh7 cells were cultured in sodium (Na⁺) buffer, including 140 mM NaCl, 5 mM KCl, 2.5 mM CaCl₂, 1 mM MgSO₄, 1 mM KH₂PO₄, and 10 mM HEPES, or in sodium-free (Na⁺-free) buffer, including 140 mM N-methyld-glucamine instead of NaCl, for 1 hour [22 (link)]. Cells were treated with 10 nM empagliflozin and dapagliflozin (Cayman Chemical) in glucose-free DMEM low-glucose (5 mmol/L) for 24 hours to determine the change of glucose uptake by SGLT2 inhibitor in hepatocytes. Then, the cell culture medium was replaced by sodium buffer and exposed to 200 μg/mL 2-(N-(7-nitrobenz-2-oxa1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG), a fluorescently labeled deoxyglucose analog, for 1 hour, washed and imaged by fluorescence microscope (IX71, Olympus, Tokyo, Japan). Moreover, cells were lysed with 0.1 M NaOH, neutralized with 0.1 M HCl, and measured using a microplate reader (excitation/emission=485/535 nm).

Lee J., Hong S.W., Kim M.J., Lim Y.M., Moon S.J., Kwon H., Park S.E., Rhee E.J, & Lee W.Y. (2024). Inhibition of Sodium-Glucose Cotransporter-2 during Serum Deprivation Increases Hepatic Gluconeogenesis via the AMPK/AKT/FOXO Signaling Pathway. Endocrinology and Metabolism, 39(1), 98-108.

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Synthesis and Procurement of SGLT2 Inhibitors

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Canagliflozin and TA-1887 were synthesized by Mitsubishi Tanabe Pharma Corporation^{27 (link),36 (link)}. Dapagliflozin and sotagliflozin were purchased from Cayman Chemical Company. Phlorizin, phloretin and α-MG were purchased from Sigma-Aldrich.

Hiraizumi M., Akashi T., Murasaki K., Kishida H., Kumanomidou T., Torimoto N., Nureki O, & Miyaguchi I. (2023). Transport and inhibition mechanism of the human SGLT2–MAP17 glucose transporter. Nature Structural & Molecular Biology, 31(1), 159-169.

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Synthesis of Dapagliflozin, a Potent SGLT2 Inhibitor

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Dapagliflozin ((1S)-1, 5-anhydro-1-C-[4-chloro-3-[(4-ethoxyphenyl) methyl] phenyl]-D-glucitol) was synthesized by Cayman Chemical (MI, USA) at an estimated purity of >98%.

Chiba Y., Yamada T., Tsukita S., Takahashi K., Munakata Y., Shirai Y., Kodama S., Asai Y., Sugisawa T., Uno K., Sawada S., Imai J., Nakamura K, & Katagiri H. (2016). Dapagliflozin, a Sodium-Glucose Co-Transporter 2 Inhibitor, Acutely Reduces Energy Expenditure in BAT via Neural Signals in Mice. PLoS ONE, 11(3), e0150756.

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Glucose and SGLT2i Regulate S6 Phosphorylation

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LLC-PK1 and HK-2 cells were cultured in DMEM supplemented with 10% fetal bovine serum and 100 IU/mL penicillin/streptomycin (Biological Industries) at 37 °C in a humid atmosphere with 5% CO₂. For studying S6 phosphorylation, cells were starved in serum-free DMEM for 1 h and then exposed to either 5 mM or 30 mM D-glucose (low and high glucose, respectively) in the presence or absence of SGLT2i (dapagliflozin; 5 μM, Cayman Chemical) for 0.5 h. For immunofluorescence, cells were exposed to either 5 mM or 30 mM D-glucose in the presence or absence of SGLT2i (5 μM) for 48 h. Rapamycin and Torin-1 (Cell Signaling) were added to the medium at a concentration of 100 and 250 nM, respectively. For inhibiting system L amino acid transporters, cells were treated with 20 mM BCH (Sigma-Aldrich) for 24 h.
For immunofluorescence, cells were grown on glass coverslips and fixed with 4% paraformaldehyde. After permeabilization and blocking, cells were incubated with anti-pS6 antibodies (5364S, Cell Signaling) followed by incubation with Cy3-conjugated anti-rabbit secondary antibodies (Jackson ImmunoResearch Laboratories). Cells were mounted on slides and examined with a Nikon A1R confocal microscope.

Kogot-Levin A., Hinden L., Riahi Y., Israeli T., Tirosh B., Cerasi E., Mizrachi E.B., Tam J., Mosenzon O, & Leibowitz G. (2020). Proximal Tubule mTORC1 Is a Central Player in the Pathophysiology of Diabetic Nephropathy and Its Correction by SGLT2 Inhibitors. Cell Reports, 32(4), 107954.

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Cardiac Fibroblast Cell Cultivation

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Materials. The CS4Z055R (containing serum) and CS4Z3500R (not containing serum) media were purchased from Cell Systems Corporation, (Kirkland, WA, USA). D-(+)-Glucose, D-(+)-mannitol, and diamidino-2-phenylindole (DAPI) were purchased from Nacalai Tesque Inc., (Kyoto, Japan). Phlorizin and dapagliflozin were purchased from Cayman Chemical Company, (Ann Arbor, MI, USA). All other chemicals were of reagent grade and commercially available.
Cell culture. Primary human cardiac fibroblast cells (ACBRI 5118) at passage 2 were purchased from Cell Systems Corporation. All studies were performed with HCFs at passage 4-10. Passage Reagent Group (Cell Systems Corporation) were used for cell passaging. The cells were seeded on 6-well tissue culture plates at a density of 1x10 5 cells/well, maintained in the CS4Z055R medium, and grown in the cell incubator at 37˚C, containing 95% O 2 and 5% CO 2 . After sub-confluence, CS4Z3500R (without serum) medium was used to synchronize the cells for 24 h. The cells were washed twice with phosphate-buffered saline and cultured in the medium to be further subjected to different treatments. Cells were passaged in 0.05% trypsin-EDTA.

Meng L., Uzui H., Guo H, & Tada H. (2018). Role of SGLT1 in high glucose level-induced MMP-2 expression in human cardiac fibroblasts. Molecular medicine reports, 17(5).

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