SOD activity was monitored according to Almeida et al. [100 (link)], using the cytochrome C method, with xanthine/xanthine oxidase as the source of superoxide radicals. A reaction solution containing 50 mM potassium phosphate buffer with 1 mM Na-EDTA (Sigma-Aldrich) (pH 7.8), 0.7 mM xanthine (Sigma-Aldrich), 0.03 mM cytochrome C (Sigma-Aldrich), 0.1 mM Na-EDTA, and 0.03 U/mL xanthine oxidase (Sigma-Aldrich) was added to previously diluted samples (0.3 mg/mL total protein content) in triplicate wells of a microplate. The absorbance was read at 550 nm for 3 min in intervals of 20 s. Activity is reported in units of SOD per mg of protein. One unit of activity was defined as the amount of enzyme necessary to produce a 50% inhibition of the cytochrome C reduction rate.
Na edta
Manufactured by Merck Group
Sourced in United States
Na-EDTA is a sodium salt of ethylenediaminetetraacetic acid (EDTA), a chelating agent. It is commonly used in analytical laboratories and scientific research as a component in sample preparation and analysis procedures.
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Lab products found in correlation
Ascorbic acid, by Merck Group (2 mentions)
Bovine gelatin, by Merck Group (1 mentions)
Fibronectin, by Merck Group (1 mentions)
F2442, by Merck Group (1 mentions)
Trypsin, by Merck Group (1 mentions)
Norepinephrine, by Merck Group (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
Claycomb medium, by Merck Group (1 mentions)
Zn no3 2 6h2o, by Thermo Fisher Scientific (1 mentions)
Cr no3 3 9h2o, by Thermo Fisher Scientific (1 mentions)
Hydrochloric acid (hcl), by Chempur (1 mentions)
H2so4, by Chempur (1 mentions)
Ch3cooh, by Chempur (1 mentions)
Methyl alcohol, by Merck Group (1 mentions)
Co no3 2 6h2o, by Merck Group (1 mentions)
Pe annexin 5 apoptosis detection kit, by BD (1 mentions)
Cycletest plus dna reagent kit, by BD (1 mentions)
Hank s balanced salt solution, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
15 protocols using na edta
1
Superoxide Dismutase Activity Assay
2
Maintenance of HL1 Cardiac Cells
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HL1 cells were maintained according to published methods [15 (link)]. Briefly, cells were plated in T25 flasks coated with 25μg fibronectin (Sigma-Aldrich, F1141) in 2 mL 0.02% bovine gelatin (Sigma-Aldrich, G9391) in water. Cells were maintained in Claycomb Medium (Sigma-Aldrich, 51800C) supplemented with 10% FBS (Sigma-Aldrich, F2442, Batch 11A568, US origin), 100 μg/mL Penicillin-Streptomycin (Sigma-Aldrich, P4333), 0.1mM norepinephrine (Sigma-Aldrich, A0937), and 2mM L-glutamine (Sigma-Aldrich, G7513). norepinephrine stock (10mM) was made up in 30mM ascorbic acid (Sigma-Aldrich, A7506) and filtered using a 0.2um syringe filter (Gelman Sciences, 4192). Cells were grown at 37°C in 5% CO2 and were passaged upon reaching confluence every 2–3 days by dissociating into single cells using 0.05% Trypsin in 0.02% EDTA-Na (Sigma-Aldrich, T3924).
3
Immobilized Supported Liquid Membranes for Metal Ions
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Deionized water, with medium electrolytic conductivity not exceeding 1 µS/cm at 20 °C, was used for preparing aqueous solutions, along with the following compounds of analytical grade: Pb(NO3)2 (POCh, Gliwice, Poland), Zn(NO3)2 6H2O (POCh), Cd(NO3)2 4H2O (Acros Organics, Geel, Belgium), Cr(NO3)3 9H2O (Acros), HNO3 (POCh), HCl (Chempur, Piekary Slaskie, Poland), H2SO4 (POCh), CH3COOH (POCh), CH3COONa (Chempur), EDTA-Na (Sigma-Aldrich, Saint Louis, MO, USA) and 2-[4-(2-hydroxyethyl)-1- piperazinyl]-etanosulfonic acid (HEPES, Sigma-Aldrich). The following compounds were used for preparing the membranes: analytical grade trichloromethane (POCh) as the carrier solvent, o-nitrophenyl octyl ether (o-NPOE) and o-nitrophenyl pentyl ether (o-NPPE) with ≥99% purity (Fluka, Busch, Switzerland), bis(2-ethylhexyl) adipate (DOA) with 99% purity (Sigma-Aldrich) as plasticizers, cellulose triacetate (CTA, Fluka) as the polymer support and calixresorcin[4]renes with the structures shown in Figure 1 . Synthesis of the carriers was described earlier in the papers [16 (link),17 (link)].
Immobilized supported liquid membranes (SLM) were prepared from 25 µm thick Celgard 2500 type (Hoechst Celanese Corporation, Charlotte, NC, USA) of microfiltration polypropylene membrane sheets, with 45% porosity, pore curvature of 2.22 and pore diameter between 0.04 and 0.4 µm.
Immobilized supported liquid membranes (SLM) were prepared from 25 µm thick Celgard 2500 type (Hoechst Celanese Corporation, Charlotte, NC, USA) of microfiltration polypropylene membrane sheets, with 45% porosity, pore curvature of 2.22 and pore diameter between 0.04 and 0.4 µm.
4
Cytotoxicity Evaluation of Cobalt Nitrate
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Co(NO3)2·6H2O, EDTA-Na, and methyl alcohol were obtained from Sigma (St. Louis, MO, USA). 1,2-diaminobenzene (OPD) and hydrogen peroxide (H2O2, 30%) were obtained from J&K China Chemical Ltd. (Shanghai, China). A 2′,7′-dichlorofluorescin diacetate (DCFH-DA) Kit and Live/Dead Cell Staining Kit (Calcein-AM/PI) were obtained from Sigma. Cells were purchased from the national collection of authenticated cell cultures. A CycleTEST™ Plus DNA Reagent Kit and PE Annexin V Apoptosis Detection Kit were obtained from BD Biosciences (Franklin Lakes, NJ, USA). The BALB/c mice (4~5 weeks old, 18~20 g) were obtained from Hualan Biology. The animal experiments were approved by the Use Committees of Henan Normal University.
5
Alkaline Comet Assay for Genotoxicity
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The alkaline (pH.13) comet assay for quantifying DNA lesions was conducted following the protocol used in the international validation of the in vivo rodent alkaline comet assay for detecting genotoxic carcinogens. Rabbit livers were collected and minced using scissors in cold mincing buffer. The mincing buffer consisted of Mg++ and Ca++ free Hank’s Balanced Salt Solution (Thermo Fisher Scientific, Waltham, MA, USA) with 20 mM Na EDTA (Sigma-Aldrich Co.) and 10% v/v dimethyl-sulfoxide (Sigma-Aldrich Co.). The comet assay protocol has been previously described.30 (link)
6
Quantification of Serotonin Levels in Ileum
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On the sacrifice day, 2–3 cm of the ileum were rapidly dissected, snap-frozen (dry ice) and stored at -80°C until use. Perchloic acid (0.2 N) was added to each sample and, after disruption and sonication (1 min on ice), samples were centrifuged at 4°C at 5000 rpm. The supernatant was filtered through a Spin-X high-performance liquid chromatography (HPLC) column (Costar, Lowell, MA, USA) to remove debris. Levels of 5-HT was measured by HPLC combined with electrochemical detection using a Gilson instrument (Gilson, Middleton, WI, USA), fitted with an analytical column (Supelco Supelcosil LC-18 3μM, flow rate: 1.0 ml/min, Bellefonte, PA, USA) as previously described [31 (link)]. Briefly, 150μl supernatant aliquots were injected into the system, using a mobile phase of 0.7 M aqueous potassium phosphate (pH 3.0) in 10% methanol, 1-heptanesulfonic acid (222mg l-1, Sigma-Aldrich, Saint Louis, MO, USA) and Na-EDTA (40 mg l-1, Sigma-Aldrich, Saint Louis, MO, USA). A standard curve using known concentrations of serotonin was run each time.
7
Cardiovascular and Renal Assessment of Doxorubicin-Induced Toxicity in Rats
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In the functional and morphological study (exp. 1), rats were anesthetized with ketamine + xylazine 8 weeks after DXR administration. To prevent blood clotting, 1 ml/kg Na-EDTA (Sigma-Aldrich Corporation, Saint Louis, MO, USA) was injected intraperitoneally. Rats were bled from the aorto-femoral bifurcation. Animals were perfused through the aorta with 60 ml cold physiological saline to remove blood from the vasculature. After perfusion, the left kidney and the heart were removed and sectioned for further analysis. The heart and a third of the left kidney were fixed in 4% buffered formaldehyde and were later embedded in paraffin for basic histological and immunohistochemical analysis. The remaining two third of the left kidney cortex and medulla were separated, frozen in liquid nitrogen and stored at -80°C for molecular studies.
8
Ultrastructural Analysis of Temporal Bone
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After decapitation, the temporal bones were stored with 2.5% glutaraldehyde in a 0.1 M phosphate buffer for 12 hours, and then rinsed with 0.1 mL/L phosphate buffer at pH 7.4 for 1 day. After incubation in 0.1 M disodium ethylenediaminetetraacetic (Na-EDTA, Sigma-Aldrich, St. Louis, MO, US) decalcifier (pH: 7.4) for 3 weeks, we opened the tympanic bulla of the temporal bones, and the otic capsule of the cochlea was removed under stereo microscopy (Olympus 1 × 71 S8 F3, Olympus Corporation, Shinjuku, Tokio, Japan). The cochleae were post-fixed with 1% osmium tetroxide in phosphate buffer for 1 hour and then rinsed with phosphate buffer. The tissues were dehydrated through a graded series of ethanol. Subsequently, in order to define the cut-off areas of the blocks, semi-thin sections and toward basal to apex were vertically obtained with an RMC-MTX Ultramicrotome (Boeckeler Instruments, Inc., Tucson, AR, US), and stained with azure blue. Thin sections with a thickness of 40–60 nm from the determined areas were obtained and stained with uranyl acetate and Reynold's lead citrate to increase contrast. Those sections were evaluated and photographed using a transmission electron microscope (JEOL 1010, JEOL, Akishima, Tokyo, Japan).
9
Western blot analysis of PDCD4 and GAPDH
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Frozen heart tissue was homogenized in lysis buffer (20 mM Tris HCl (Sigma-Aldrich, Germany), 150 mM NaCl (Roth, Germany), 1 mM Na-EDTA (Sigma-Aldrich, Germany), 1 mM EGTA (Roth, Germany), 1% NP40, 2.5 mM sodium pyruvate (Sigma-Aldrich, Germany), 2.5 mM sodium vanadate (Sigma-Aldrich, Germany) and freshly added protease inhibitor mix (Complete; Roche, Germany). After determination of protein concentration via the Lowry method [19 (link)] the western blot analysis was performed, as previously described in detail [16 (link)]. As primary antibodies were used: rabbit anti PDCD4 (#9535, cell signaling, 1:1000) and mouse anti GAPDH (ab8245, abcam, 1:40,000). For detection chemiluminescence was used via the following secondary antibodies purchased from Jackson ImmunoResearch Laboratories Inc.: Peroxidase AffiniPure Donkey Anti-Rabbit IgG (H + L) (#711-035-152, 1:10,000) and Peroxidase AffiniPure Goat Anti-Mouse IgG (H + L) (115-035-003, 1:10,000).
10
Trace Metal Analysis by IC-ICP-MS
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Ultrapure water (<0.005 µS) obtained from a Milli-Q Direct 8 purification unit (Millipore, Burlington, MA, USA, Merck) was used to prepare all the solutions. Ammonium nitrate, potassium dichromate, standard solution of Cr(III) and Cr(VI)—1000 mg/L (Merck, USA), Na-EDTA, diluted hydrochloric acid, and ammonia (Sigma−Aldrich, St. Louis, MI, USA) to adjust the pH of the mobile phase were used for the analysis. All standard solutions used for the calibration process were prepared by volume dilution of Cr standard solution 1000 mg/L. To avoid contamination, all glassware and storage bottles were kept in 10% (v/v) nitric acid for at least 48 h, rinsed three times with ultrapure water, and preserved dried till use.
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