Wild type SIRT2 (SIRT2pcDNA3.1; Plasmid number 13813) was obtained from Addgene and pcDNA 3.1 was purchased from ThermoFisher Scientific. SH-SY5Y cells were seeded in 12-well plates and the cells were transfected with SIRT2pcDNA3.1 and the control group was transfected with empty pcDNA3.1 plasmid using PEI (polyethyleneimine; Invitrogen). Plasmids were incubated with cells at 37°C for 48 hours. To study the effect of SIRT2 inhibition, one set of cells transfected with SIRT2 and pcDNA3.1 were treated with either diquat (Sigma-Aldrich, UK) dissolved in PBS (Phosphate buffered saline; Sigma-Aldrich) or rotenone (Sigma-Aldrich) dissolved in DMSO (dimethyl sulphoxide, Sigma-Aldrich) at a final concentration of 0.2% PBS/DMSO alone and a second set of cells with toxin and AGK2 as a specific SIRT2 inhibitor (25 μM; Tocris, UK; see Supplementary Figure 4 in Supplementary Material available online at https://doi.org/10.1155/2017/2643587 ). AGK2 was added to cells 2 hours prior to diquat or rotenone treatment and the cells were incubated overnight for 20 hours. Cell viability was determined by Alamar Blue reduction assay [20 (link)] (refer to Figures 1, 2, and 4 in supplementary files for efficiency of SIRT2 transfection and inhibition).
Phosphate buffered saline (pbs)
Manufactured by Bio-Techne
Sourced in United Kingdom, United States
PBS (Phosphate-Buffered Saline) is a commonly used buffer solution that provides a physiologically compatible environment for biological samples. It is a balanced salt solution designed to maintain pH and osmolarity, making it suitable for various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Diquat, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Bio-Techne (1 mentions)
Rotenone, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Takara Bio (1 mentions)
Proteoguard edta free protease inhibitor cocktail, by Takara Bio (1 mentions)
Fetal bovine serum (fbs), by American Type Culture Collection (1 mentions)
Cisplatin, by Cayman Chemical (1 mentions)
Cisplatin, by Bio-Techne (1 mentions)
Human umbilical vein endothelial cells huvecs, by ScienCell (1 mentions)
Acutase cell detachment solution, by BD (1 mentions)
Annexin 5 fluorescein isothiocyanate fitc apoptosis detection kit 1, by BD (1 mentions)
Vegf165, by Merck Group (1 mentions)
Il 1β, by Merck Group (1 mentions)
Propidium iodide, by BD (1 mentions)
Silica gel 60, by Merck Group (1 mentions)
Methanol, by Merck Group (1 mentions)
Transwell insert, by Corning (1 mentions)
Tlc silica gel 60 f254, by Merck Group (1 mentions)
N hexane, by Merck Group (1 mentions)
Calcein am, by BD (1 mentions)
7 protocols using phosphate buffered saline (pbs)
1
SIRT2 Overexpression and Inhibition in SH-SY5Y Cells
2
Hypoxia Mimetic and Acetazolamide in Mice
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For the hypoxia mimetic study, 1 mM dimethyloxalylglycine (DMOG, in PBS, Tocris, 22 mice) or vehicle (16 mice) was intrathecally injected into C57Bl6 adult male mice (total 38) under recovery anaesthesia (isoflurane ∼2% oxygen). Acetazolamide (ACZ) was delivered through intraperitoneal injection (40 mg/kg, in 1% DMSO in PBS, Tocris).
3
Metabolic Effects of Spexin Treatment in Mice
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Normal adult male C57BL/6 mice (four months of age) were kept in a temperature-controlled environment (20–22 °C and a fixed 12 h light/12 h dark cycle; lights on at 07:00 h) and fed ad libitum with Purina commercial rat/mouse chow (Ganave, Buenos Aires, Argentina, L0723/1). Mice were injected with SPX or PBS (29 µg/kg/day, Tocris, Bristol, UK) for ten days (the SPX and CTR groups, respectively). On the third day of the protocol, each group was subdivided into two other groups: one exposed to cold (4 °C, CTR-C and SPX-C, n = 12 mice for each group) and another one housed at RT (CTR and SPX, n = 15 mice for each group) (Figure 1 ). Food intake and body weight were measured every day. On the experimental day (day 10), the mice were euthanized under non-fasting conditions (08:00 to 09:00 h), and their trunk blood was collected; plasma samples were then frozen (–80 °C) until the metabolite measurement. Inguinal AT (IAT, subcutaneous depot), epididymal AT (EAT, visceral depot), and brown AT (BAT, subcutaneous depot) were aseptically dissected and weighed. These depots were kept at –80 °C for further procedures. Animals were euthanized according to protocols for animal use, in agreement with the NIH guidelines for the care and use of experimental animals. All experiments were approved by our Institutional Animal Care Committee (Approval code: 6NE-2023).
4
Preparation of HepG2 Cell Lysates
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Reagents and medium were purchased from Sigma-Aldrich (Sant Louis, MO, USA), unless otherwise noted. PBS was purchased from Trevigen (Gaithersburg, MD, USA) and supplemented with 10 µL of ProteoGuard™ EDTA-Free Protease Inhibitor Cocktail (Takara Bio USA, Inc., Mountain View, CA, USA) per 1 mL. HepG2 cells were grown in EMEM medium supplemented with 8% fetal bovine serum (ATTC), 1675 mM L-glutamine, 85 U/mL penicillin, 85 μg/mL streptomycin of 80% confluence. Cells were harvested and centrifugated at 340 g for 2 min at 4 °C and resuspended in 50 mL PBS. After a second wash step, the cells were resuspended in 10 mL ice-cold PBS and centrifugated again at 340 g for 2 min at 4 °C. Washed pellets were either used directly or snap frozen in liquid nitrogen and stored at −80 °C until lysis.
5
HepG2 Cell Harvesting and Lysis Protocol
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Reagents and medium were purchased from Sigma-Aldrich (Sant Louis, MO, USA) unless otherwise noted. PBS was purchased from Trevigen (Gaithersburg, MD, USA). HepG2 cells were grown until 70% confluence in EMEM medium supplemented with 7% fetal bovine serum (ATCC, Manassas, VA, USA), 1675 mM L-glutamine, 85 U/mL penicillin, and 85 µg/mL streptomycin. Cells were harvested and centrifugated at 340× g for 4 min at 4 °C. Three washes were made with 30 mL of cold PBS. Resuspension and centrifugation at 340× g for 4 min at 4 °C. Washed pellets were snap-frozen in liquid nitrogen and stored at −80 °C until lysis.
6
Cisplatin-Induced Nephrotoxicity in Mice
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Seven-week-old male C57BL/6 mice were purchased from HyoSung Science (Daegu, Republic of Korea). Before starting experiments, the mice were acclimated for 1 week under 20–24 °C on a 12/12 h light/dark cycle. Animal experiments were approved by the Institutional Animal Care and Use Committee of the Daegu Catholic University Medical Center (DCIAFCR-180312-20-Y). The mice were randomly grouped into four groups (n = 8 in each group): (1) control (Con) group; (2) OXA group; (3) cisplatin (CP) group; (4) cisplatin plus OXA (CP + OXA) group. The CP group and the CP + OXA group were injected intraperitoneally with cisplatin [15 mg/kg in 0.9% saline; Cayman Chemical, Ann Arbor, MI, USA]. The OXA group and the CP + OXA group were given an intraperitoneal injection of OXA (1 μmol/kg in phosphate-buffered saline (PBS); Tocris Bioscience, Bristol, UK) daily for 4 consecutive days, starting from 1 day prior to 0.9% saline or cisplatin injection. An equal volume of PBS was injected intraperitoneally into the CP group. The doses of cisplatin and OXA were chosen based on previous studies [16 (link),17 (link),18 (link),19 (link)]. At 72 h after cisplatin injection, the mice were sacrificed.
7
HUVEC and HFLS-RA Co-culture Assay
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Human umbilical vein endothelial cells (HUVECs) and endothelial cell medium (ECM) were purchased from ScienCell, USA; Human fibroblast-like synoviocytes for rheumatoid arthritis (HFLS-RA) and synoviocytes growth medium (SGM) were supplied by Cell Applications INC (USA); VEGF165 and IL-1β were purchased from Sigma-Aldrich, USA; Suramin hexasodium salt and dimethyl sulfoxide (DMSO) were obtained from Abcam, UK; 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliumbromide (MTT) reagent and phosphate buffered saline (PBS) were purchased from Tocris Bioscience, UK; Matrigel™ (10 mg/mL) and Transwell inserts were obtained from Corning, USA; Ethanol, n-hexane, methanol, ethyl acetate, Silica gel 60 (0.063–0.200 mm), and thin-layer chromatography (TLC Silica gel 60 F254) were supplied from Merck, Germany; Whatman filter paper (No. 1) was supplied by Whatman Ltd., England. Hematoxylin and eosin (H & E) stain was supplied from Cellpath, UK; Fluorescein isothiocyanate (FITC) Annexin V Apoptosis Detection Kit I, Acutase™ cell detachment solution, propidium iodide, and calcein AM were purchased from BD Pharmingen™, USA.
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