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Detapac

Manufactured by Merck Group
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Sourced in United States, Poland

DETAPAC is a lab equipment product manufactured by Merck Group. It is a device used for the detection and analysis of various compounds and materials. The core function of DETAPAC is to perform analytical tasks in a laboratory setting.

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Lab products found in correlation

Xanthine, by Merck Group (3 mentions) Bathocuproinedisulfonic acid, by Merck Group (2 mentions) Catalase, by Merck Group (2 mentions) Xanthine oxidase, by Merck Group (2 mentions) Sodium phosphate, by Merck Group (2 mentions) 5 sulfosalicylic acid, by Merck Group (2 mentions) Glutathione reductase, by Merck Group (2 mentions) Ethylene diamine tetraacetic acid (edta), by Merck Group (2 mentions) Xmark microplate absorbance spectrophotometer, by Bio-Rad (2 mentions) Deferoxamine, by Merck Group (1 mentions) Doxycycline, by Thermo Fisher Scientific (1 mentions) Bovine catalase, by Merck Group (1 mentions) Glutathione disulfide, by Merck Group (1 mentions) 1 octanesulfonic acid, by Merck Group (1 mentions) Glutathione (gsh), by Merck Group (1 mentions) Perchloric acid, by Merck Group (1 mentions) 5 5 dithiobis 2 nitrobenzoic acid, by Merck Group (1 mentions) 5 5 dithiobis 2 nitrobenzoic acid dtnb, by Merck Group (1 mentions) Nadph, by Merck Group (1 mentions) Haemoglobin, by Merck Group (1 mentions)

9 protocols using detapac

1

Iron Chelation and Antioxidant Assay

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Drugs were added to cells at final concentrations of 250 μM iron sucrose (Venofer, American Regents Inc.), 2.5–10 mM ascorbate (Macron Chemicals) that was prepared in sodium bicarbonate to maintain a neutral pH, 20–200 μM deferoxamine (Sigma), 1 mM diethylenetriaminepentaacetic acid (DETAPAC) (Sigma), 100 Units/mL bovine catalase (Sigma), and 0.5–2 μg/mL doxycycline (Fisher Scientific).
Brandt K.E., Falls K.C., Schoenfeld J.D., Rodman S.N., Gu Z., Zhan F., Cullen J.J., Wagner B.A., Buettner G.R., Allen B.G., Berg D.J., Spitz D.R, & Fath M.A. (2017). Augmentation of intracellular iron using iron sucrose enhances the toxicity of pharmacological ascorbate in colon cancer cells. Redox Biology, 14, 82-87.
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2

Spectrophotometric Assay of Superoxide Dismutase

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The measurement of SOD activity is based on the inhibition of nitroblue tetrazolium (NBT) reduction by O2•− [47 ,48 (link)]. Pancreas homogenates were incubated in 50 mM potassium phosphate buffer (pH 7.8) containing 1 mM diethylen-etriaminepentaacetic acid (DETAPAC) (Sigma Aldrich, St. Louis, MO #D6518), 0.13% BSA, 1 unit/ml catalase (Sigma Aldrich, St. Louis, MO, #C-40), 56 μM NBT (Sigma Aldrich St. Louis, MO, #N6876), 0.1 mM xanthine (Sigma Aldrich St. Louis, MO, #X0125), and 50 μM bathocuproine disulfonic acid (Sigma Aldrich, St. Louis, MO, #146625) for 30 min. xanthine oxidase (Sigma Aldrich St. Louis, MO, #X187) was added to give an NBT reduction rate of 0.015–0.025/min at 560 nm. One minute after sample solution was mixed with 0.01 units/ml xanthine oxidase, the absorbance was measured at 560 nm for 2 min at 15 s intervals. To assay MnSOD, 5 mM NaCN (Sigma Aldrich, St. Louis, MO, #380970) was added to the buffer and allowed to incubate for 1 hr. One unit of SOD activity was defined as the quantity of protein required for half of the maximal inhibition of NBT reduction.
O’Malley Y., Coleman M.C., Sun X., Lei J., Yao J., Pulliam C.F., Kluz P., McCormick M.L., Yi Y., Imai Y., Engelhardt J.F., Norris A.W., Spitz D.R, & Uc A. (2022). Oxidative stress and impaired insulin secretion in cystic fibrosis pig pancreas. Advances in redox research : an official journal of the Society for Redox Biology and Medicine and the Society for Free Radical Research-Europe, 5, 100040.
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3

Quantification of Cellular Antioxidants

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Cells grown in 100 mm2 dishes were treated with IR, ascorbate, or in combination. After treatments, cells were washed with PBS and harvested by trypsinization (floating cells in culture media and PBS were collected and combined with trypsinized cells). After centrifugation, cell pellets were suspended in 100 μL PCA buffer (5% perchloric acid with 100 μM DETAPAC (diethylenetriaminepentaacetic acid); Sigma-Aldrich Chemical Co., St. Louis, MO) to precipitate protein. Samples were sonicated and centrifuged at 18,500 g for 5 min at 4 ° C using an Eppendorf Microcentrifuge. Supernatants were collected and stored at −80 ° C or analyzed immediately using HPLC (ESA CoulArray, Dionex/Thermo Scientific, Sunnyvale, CA) with electrochemical detection (ECD) following the protocol described in Park et al. (18 (link)). In addition, in separate groups of treated mice, red blood cells (RBCs) were harvested and centrifuged (500 g for 5 min). The plasma was removed from the sample and the remaining pellet of intact RBCs was washed twice with cold isotonic saline. An aliquot was removed for counting by a hemocytometer. From the RBC pellet, 100 μL of RBCs were lysed with 300 μL of PCA buffer and then centrifuged to pellet the protein (500 g, 5 min). The clean supernatant was stored at −80 ° C or immediately analyzed for total glutathione (tGSH) using a plate-reader based assay (19 (link)).
Du J., Cieslak JA I.I.I., Welsh J.L., Sibenaller Z.A., Allen B.G., Wagner B.A., Kalen A.L., Doskey C.M., Strother R.K., Button A.M., Mott S.L., Smith B., Tsai S., Mezhir J., Goswami P.C., Spitz D.R., Buettner G.R, & Cullen J.J. (2015). Pharmacological Ascorbate Radiosensitizes Pancreatic Cancer. Cancer research, 75(16), 3314-3326.
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4

Trace Element Quantification Protocol

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Perchloric acid, glutathione, glutathione disulfide, 1-octanesulfonic acid, sodium mono-phosphate, and diethylenetriaminetetraacetic acid (DTPA or DETAPAC) were from Sigma Aldrich (St. Louis, MO, USA). CELLPACK™ analyzer solution was obtained from Sysmex Corporation, Kobe, Japan.
van ‘t Erve T.J., Doskey C.M., Wagner B.A., Hess J.R., Darbro B.W., Ryckman K.K., Murray J.C., Raife T.J, & Buettner G.R. (2014). The Heritability of Glutathione and Related Metabolites in Stored Red Blood Cells. Free radical biology & medicine, 0, 107-113.
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5

Spectrophotometric Assay of Superoxide Dismutase

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The measurement of SOD activity is based on the inhibition of nitroblue tetrazolium (NBT) reduction by O2•− [47 ,48 (link)]. Pancreas homogenates were incubated in 50 mM potassium phosphate buffer (pH 7.8) containing 1 mM diethylen-etriaminepentaacetic acid (DETAPAC) (Sigma Aldrich, St. Louis, MO #D6518), 0.13% BSA, 1 unit/ml catalase (Sigma Aldrich, St. Louis, MO, #C-40), 56 μM NBT (Sigma Aldrich St. Louis, MO, #N6876), 0.1 mM xanthine (Sigma Aldrich St. Louis, MO, #X0125), and 50 μM bathocuproine disulfonic acid (Sigma Aldrich, St. Louis, MO, #146625) for 30 min. xanthine oxidase (Sigma Aldrich St. Louis, MO, #X187) was added to give an NBT reduction rate of 0.015–0.025/min at 560 nm. One minute after sample solution was mixed with 0.01 units/ml xanthine oxidase, the absorbance was measured at 560 nm for 2 min at 15 s intervals. To assay MnSOD, 5 mM NaCN (Sigma Aldrich, St. Louis, MO, #380970) was added to the buffer and allowed to incubate for 1 hr. One unit of SOD activity was defined as the quantity of protein required for half of the maximal inhibition of NBT reduction.
O’Malley Y., Coleman M.C., Sun X., Lei J., Yao J., Pulliam C.F., Kluz P., McCormick M.L., Yi Y., Imai Y., Engelhardt J.F., Norris A.W., Spitz D.R, & Uc A. (2022). Oxidative stress and impaired insulin secretion in cystic fibrosis pig pancreas. Advances in redox research : an official journal of the Society for Redox Biology and Medicine and the Society for Free Radical Research-Europe, 5, 100040.
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6

Glutathione Reductase Assay for Cell Lysates

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One million cells were lysed in 1.34mM Diethylenetriaminepenta-acetic acid (DETAPAC, Sigma) dissolved in 143 mM sodium phosphate (Sigma), 6.3 mM EDTA (Sigma), and 5% 5-sulfosalicylic acid (SSA, Sigma). 50 μL of lysate were mixed with 700 μL 0.298 mM NADPH (Sigma) in sodium phosphate buffer, 100 μL 6mM 5,5′-dithio-bis-2-nitrobenzoic acid (DTNB, Sigma) sodium phosphate buffer, 100 μL water (Corning), and 50 μL 0.023 U/μL glutathione reductase (GR) (Sigma). The kinetic absorbance was read at 412 nm every 15 s for 2.5 min using xMark™ Microplate Absorbance Spectrophotometer (BioRad), and the rates were compared to a standard curve. Tumors were chopped and lysed in DETAPAC buffer before assayed. Protein concentrations were measured for standardization of total glutathione levels.
Zou X., Zhu Y., Park S.H., Liu G., O’Brien J., Jiang H, & Gius D. (2017). SIRT3-mediated dimerization of IDH2 directs cancer cell metabolism and tumor growth. Cancer research, 77(15), 3990-3999.
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7

EPR Assay for Superoxide Radical Scavenging

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On a JEOL JES300ESR, the signal of ·O2− 2 free radicals, which were produced by the reaction of 5 μL of the solution of 11.316 mg of dimethylpyridine N-oxide (DMPO; Sigma-Aldrich Co.) in 1 mL of ultrapure water (0.1 M), 5 μL of a solution of diethylenetriamine pentaacetic acid (DETAPAC; Sigma-Aldrich Co.) in ultrapure water (0.9 mM), 5 μL of the solution of 0.3 g of xanthine (Sigma-Aldrich Co.) in 1 mL of ultrapure water (0.5 M), and 5 μL of the solution of commercial xanthine oxidase (Sigma-Aldrich Co.) diluted by ultrapure water (1/10, v/v) was measured; the height of the signal was calculated and defined as the blank height of the ·O2−signal (BH·O2−). The effect of PZL318 on the level of ·O2− free radicals was measured by the signal of ·O2− free radicals formed from 5 μL of the solution of 11.316 mg of DMPO in 1 mL of ultrapure water (0.1 M), 5 μL of a solution of DETAPAC in ultrapure water (0.9 mM), 5 μL of the solution of 0.3 g of xanthine in 1 mL of ultrapure water (0.5 M), 5 μL of the solution of commercial xanthine oxidase diluted by ultrapure water (1/10, v/v), and 5 μL of the solution of PZL318 in 1 mL of ultrapure water (final concentration: 1×10−6 M); the height of the signal was calculated and defined as the PZL318-treated height of the O2− signal (TH O2−). The O2− scavenging ratio was calculated according to the formula:
Scavengingratio=(BH⋅O2−−TH⋅O2−)/BH⋅O2−.
Li S., Wang Y., Wang F., Wang Y., Zhang X., Zhao M., Feng Q., Wu J., Zhao S., Wu W, & Peng S. (2015). Small molecule PZL318: forming fluorescent nanoparticles capable of tracing their interactions with cancer cells and activated platelets, slowing tumor growth and inhibiting thrombosis. International Journal of Nanomedicine, 10, 5273-5292.
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8

Glutathione Quantification in Cell Lysates

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One-million cells at 70–80% confluency were lysed in 1.34 mM diethylenetriaminepenta-acetic acid (DETAPAC, Sigma) and dissolved in 143 mM sodium phosphate (Sigma). Then 6.3 mM EDTA (Sigma) and 5% 5-sulfosalicylic acid (Sigma) were added to the lysates. Fifty microliters of lysate were mixed with 700 μL 0.298 mM NADPH (Sigma) dissolved in sodium phosphate buffer, 100 μL 6 mM 5,5’-dithio-bis-2-nitrobenzoic acid (DTNB, Sigma) in sodium phosphate buffer, 100 μL water, and 50 μL 0.023 U/μL glutathione reductase (GR) dissolved in water (Sigma). Kinetic absorbance was read at 412 nm every 15 s for 2.5 min using an xMark™ microplate absorbance spectrophotometer (BioRad), and the rates were compared to a standard curve. Tumors were lysed in DETAPAC buffer before being assayed, and protein concentrations were measured for standardization of GSH levels that were normalized using the BCA method.
Zhu Y., Zou X., Dean A.E., Brien J.O., Gao Y., Tran E.L., Park S.H., Liu G., Kieffer M.B., Jiang H., Stauffer M.E., Hart R., Quan S., Satchell K.J., Horikoshi N., Bonini M, & Gius D. (2019). Lysine 68 acetylation directs MnSOD as a tetrameric detoxification complex versus a monomeric tumor promoter. Nature Communications, 10, 2399.
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9

Antioxidant and Enzymatic Assays

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Ferrozine (3-(2-pyridyl)-5,6-bis-(4-phenyl-sulfonic acid)-1,2,4-triazine), ABTS (2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)), NBT (nitro blue tetrazolium), DETAPAC (diethylenetriaminepentaacetic acid), α-amylase, pancreatin, pepsin, bile extract, Folin-Ciocalteu reagent, linoleic acid, ammonium thiocyanate, and haemoglobin were purchased from Sigma-Aldrich company (Poznan, Poland). All other chemicals were of analytical grade.
Gawlik-Dziki U., Świeca M., Dziki D., Sęczyk Ł., Złotek U., Różyło R., Kaszuba K., Ryszawy D, & Czyż J. (2014). Anticancer and Antioxidant Activity of Bread Enriched with Broccoli Sprouts. BioMed Research International, 2014, 608053.
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