ARPE-19 cells (ATCC® CRL-2302™) were cultured in T25 flasks with DMEM/F12 medium for 24 h in a 5% CO2 incubator at 37 °C. The cells were detached and seeded in 96-well plates (5 × 104 cells/well) for 24 h then treated with H2O2, TG, TUDCA, H2O2 plus TUDCA, or TG plus TUDCA for 24 h. H2O2, at a concentration of 750 µM, was chosen for this study, based on our previous publication [15 (link),16 (link)]; and 1 µM TG (Cat. T9033, Sigma, Dorset, UK) is commonly used to induce ER stress in vitro [17 (link),18 (link),19 (link)]—so we also used a dose of 1 µM for this study. TUDCA (Cat. 580549, Sigma, Dorset, UK) at different concentrations were used. Cell viability was evaluated using an MTT assay (Cat. M2128, Sigma, Dorset, UK) following the manufacturer’s protocol and percentage viability calculated according to our previous description [15 (link)].
Tudca
Manufactured by Merck Group
Sourced in United States, United Kingdom, Germany
TUDCA is a bile acid derivative produced by Merck Group. It is a white crystalline powder with a molecular formula of C26H45NO6. TUDCA acts as an osmotic agent and has been observed to have cytoprotective properties.
Automatically generated - may contain errors
Lab products found in correlation
Tunicamycin, by Merck Group (10 mentions)
4 pba, by Merck Group (7 mentions)
Thapsigargin, by Merck Group (4 mentions)
Dexamethasone (dex), by Merck Group (3 mentions)
Tcdca, by Merck Group (3 mentions)
Gcdca, by Merck Group (3 mentions)
P perk, by Cell Signaling Technology (3 mentions)
Dithiothreitol (dtt), by Merck Group (2 mentions)
Rotenone, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Keratinocyte serum free medium, by Thermo Fisher Scientific (2 mentions)
Eif2α, by Cell Signaling Technology (2 mentions)
P erk, by Cell Signaling Technology (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Deoxycholic acid, by Merck Group (2 mentions)
Hyodeoxycholic acid, by Merck Group (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
α tubulin, by Merck Group (2 mentions)
Phospho jnk, by Cell Signaling Technology (2 mentions)
Parp1, by Cell Signaling Technology (2 mentions)
71 protocols using tudca
1
ARPE-19 Cells Stress and Viability
2
Aldosterone-Induced Kidney Injury Model
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All experiments were performed in accordance with the Fudan Medical University Guide for Laboratory Animals. Eight-week-old C57BL/6J mice weighting between 25 and 30 g were purchased from the Institute of Animal Care at Fudan University and underwent a right uninephrectomy under anesthesia with sodium pentobarbital (50 mg/kg, IP). After two weeks of recovery, all mice were given drinking water containing 1% NaCl and randomly treated with one of the following for four weeks: group 1, Sham+V (0.5% ethanol subcutaneously, saline vehicle i.p., n = 6); group 2, Sham+TUDCA (0.5% ethanol subcutaneously, 250 mg/kg/d of TUDCA i.p., Sigma-Aldrich, USA, n = 6); group 3, Aldo+V (Aldo 0.75 μg/h subcutaneously, Sigma-Aldrich, USA, saline vehicle i.p., n = 6); and group 4, Aldo+TUDCA (Aldo 0.75 μg/h subcutaneously, 250 mg/kg/d of TUDCA i.p., n = 6) [10 (link)]. At the end of the experiment, the mice were anesthetized and the body and kidney weight were measured. Twenty-four-hour urine samples were collected after a 24 h acclimatization period in the metabolic cages. Urinary protein excretion was determined using enzyme-linked immunosorbent assay (ELISA) kits (Exocell). Additionally, the plasma was centrifuged for testing creatinine, urea nitrogen, IL-18, and IL-1β. Kidney samples were immediately frozen in liquid nitrogen and stored at −80°C.
3
Inducing ER Stress in Drosophila and S2R+ Cells
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TUDCA (EMD Millipore) or PBA (EMD Millipore) were added to regular Drosophila food at a final concentration of 15 and 7.5 mM. To induce ER stress in vivo, dissected larvae were exposed to HL3 solution containing 500 µM DTT (Sigma-Aldrich) for 4 h. For experiments of ER stress, S2R+ cells cultured in Schneider’s medium were treated for 24 h with 1 µM thapsigargin (Sigma-Aldrich) and 400 µM TUDCA or 15 mM PBA.
4
TUDCA Effects on Fetal Liver Cells
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TUDCA (Sigma-Aldrich) was dissolved in sterile PBS and that was intraperitoneally (IP) injected (7.5 mg/kg) into pregnant mice serially from embryonic day 10.5–14.5 of pregnancy and E14.5 embryos were harvested one hour after TUDCA injections for TPE-MI analysis. For ex vivo culture Fancd2−/− fetal liver cells were cultured for 18 h in Stem Span media, supplemented with IL-6 (500 μg), IL-3 (500 μg) and SCF (2500 μg), in 50 ml Stem Span and 1% PS. For adult BM transplant experiments, recipient animals received daily serial TUDCA (IP) injections followed by two injections per week for 10 weeks at 7.5 mg/kg.
5
Optimization of Cell Culture Conditions
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D -Glucose (used at 33.3 mM) and D -ribose (50 mM) were purchased from Invitrogen (Invitrogen Corporation, Grand Island, NY, USA) and Sigma-Aldrich (St. Louis, MO, USA), respectively. Similar concentrations of L -glucose or L -ribose were used as hyperosmolarity controls (data not shown).5 (link) Thapsigargin (used at 5 μM) was purchased from Calbiochem (Darmstadt, Germany). Anisomycin (Sigma-Aldrich) was used at 1 μg/ml concentration. TUDCA (Sigma-Aldrich) was used at 0.5 mM, PBA (Sigma-Aldrich) at 2.5 mM, NAC (Sigma-Aldrich) at 1.0 mM, H2O2 (Sigma-Aldrich) at 30 μM and Rotenone (Sigma-Aldrich) at 100 nM. The concentrations of TUDCA, PBA, NAC, H2O2 and Rotenone were titrated to determine optimal culture conditions. Control conditions contained the same concentration of diluent (DMSO for Rotenone, tunicamycin and Q-VD-OPh, ethanol for thapsigargin or NaOH for PBA, other reagents were made up in complete CMRL medium) in the complete medium.
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6
Mitigating ER Stress in Trophoblast Cells
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To prevent Δ9-THC-induced ER stress, we used tauroursodeoxycholic acid (TUDCA); TUDCA has been shown to relieve ER stress in several cell and tissue types, including the placenta [35, (link)37] (link). After seeding cells in 12-well plate, cells were pretreated for 1 h with 100 μM of TUDCA (Sigma-Aldrich), and then treated with 15 μM Δ9-THC for 24 h. This dose of TUDCA has been effective to ameliorate nicotine-induced ER stress and UPR activation in rat Rcho-1 trophoblast giants cells after 24 h [35] (link). Additionally, BeWo cells were also treated with 15 μM 11-COOH-THC (Sigma-Aldrich), the inactive THC metabolic, to assess its potential effects on ER stress.
7
Immunosuppressive Treatments in NOD-Scid Mice
8
TUDCA administration in Sprague-Dawley rats
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Experimental procedures were carried out in strict accordance with the current regulations for the use of laboratory animals (ARVO statement for the use of animals in ophthalmic and visual research and European Directive 2010/63/UE) and all efforts were made to minimize animal suffering and numbers. The protocol was approved by the University of Alicante Research Ethics Committee (permit number #UA-2013-07-22). Sprague-Dawley rats, obtained from Harlan laboratories (Indianapolis, IN, USA), were used in this study. The animals were bred at the University of Alicante animal facilities and reared in an artificial 12-h light/dark cycle with food and water ad libitum.
Tauroursodeoxycholic acid (TUDCA; Calbiochem, Merck Millipore, Darmstadt, Germany) was dissolved in phosphate-buffered saline solution, pH 7.4, and sterile-filtered prior to administration. Adult (12–16 weeks) rats received a daily intraperitoneal dose of TUDCA (500 mg/kg) or vehicle (phosphate-buffered saline) for 6 days.
Tauroursodeoxycholic acid (TUDCA; Calbiochem, Merck Millipore, Darmstadt, Germany) was dissolved in phosphate-buffered saline solution, pH 7.4, and sterile-filtered prior to administration. Adult (12–16 weeks) rats received a daily intraperitoneal dose of TUDCA (500 mg/kg) or vehicle (phosphate-buffered saline) for 6 days.
9
Chaperone-Mediated Spheroid Treatment
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Two different chemical chaperones were applied in this study—tauroursodeoxycholic acid (TUDCA, Merck, Darmstadt, Germany) and sodium phenylbutyrate (4-PBA, Cayman Chemical, Ann Arbor, MI, USA). TUDCA and 4-PBA were always freshly dissolved in deionized H2O at 8 mM and 100 mM, respectively, and further diluted 1:10 in supplemented DMEM directly before use. For spheroid exposure, the supernatant was reduced to 100 µL per well and re-supplemented by 100 µL of the chaperone solution to reach final concentrations for the treatment of 0.4 mM for TUDCA and 5 mM for 4-PBA. DMEM with an equivalent H2O content was used as vehicle control. After 1 h of incubation, 50% of the chaperone-containing media were removed, plates were sealed, and the treatment procedure continued as described above. In the long-term experiments, exposure to the chaperones was terminated after 24 h by careful washing and transferring the spheroids into fresh agarose-coated 96-well plates using the automated pipetting system equipped with wide-bore tips.
10
Preparation of Chemical Reagents
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All chemicals and reagents were purchased from Sigma-Aldrich Chemical Co. (St.
Louis, MO, USA) unless otherwise indicated. Tunicamycin (TM), TUDCA (Merck,
Darmstadt, Germany), and salubrinal were dissolved in dimethyl sulfoxide (Junsei
Chemical, Tokyo, Japan) to prepare stock solutions that were then stored at
−20℃ before use.
Louis, MO, USA) unless otherwise indicated. Tunicamycin (TM), TUDCA (Merck,
Darmstadt, Germany), and salubrinal were dissolved in dimethyl sulfoxide (Junsei
Chemical, Tokyo, Japan) to prepare stock solutions that were then stored at
−20℃ before use.
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