Potassium phosphate buffer

Manufactured by Thermo Fisher Scientific

Sourced in United States

Potassium phosphate buffer is a chemical solution used to maintain a specific pH level in various laboratory applications. It is a combination of potassium dihydrogen phosphate (KH2PO4) and dipotassium hydrogen phosphate (K2HPO4) in water. The buffer helps to stabilize the pH of solutions, ensuring consistent experimental conditions.

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Lab products found in correlation

7 protocols using potassium phosphate buffer

SOD Enzyme Quantification Assay

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A standard curve was prepared using 100 units/mL of SOD enzyme solution (Sigma-Aldrich, MO, USA) as a standard compound. SOD levels were determined by adding the supernatant into the reaction cocktail (pH 7.8) containing 0.108 mM xanthine solutions (Sigma-Aldrich, MO, USA), 216 mM potassium phosphate buffer (pH 7.8, Ajax Finechem, Auckland, New Zealand), 1.1 mM cytochrome C (Sigma-Aldrich, MO, USA), and 10.7 mM ethylenediaminetatraacetic acid (Sigma-Aldrich, MO, USA). After that, the reaction mixture was reacted with 0.1 units/mL of xanthine oxidase enzyme solution (Sigma-Aldrich, MO, USA) before analyzing the absorbance at 540 nm at 0 and 5 min in triplicate.

Suwannakot K., Sritawan N., Naewla S., Aranarochana A., Sirichoat A., Pannangrong W., Wigmore P, & Welbat J.U. (2022). Melatonin Attenuates Methotrexate-Induced Reduction of Antioxidant Activity Related to Decreases of Neurogenesis in Adult Rat Hippocampus and Prefrontal Cortex. Oxidative Medicine and Cellular Longevity, 2022, 1596362.

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Spectrophotometric Assay for Catalase

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1000 units/mL of CAT enzyme solution (Sigma-Aldrich, MO, USA) was prepared as a standard compound. The supernatant was added to the solutions such as 50 mM potassium phosphate buffer, pH 7.0 (Ajax Finechem, Auckland, New Zealand), 30% hydrogen peroxide (Merck, Darmstadt, Germany), and sulfuric acid (RCI Labscan, Bangkok, Thailand). The reaction compound was incubated in the dark condition for 10 min before reacted with potassium permanganate solution (pH 7.0, Ajax Finechem, Auckland, New Zealand). The concentration of CAT was detected by evaluating the absorbance at 540 nm, and each sample was performed in triplicate.

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PM2.5 Exposure on Lung Cell Viability

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BEAS-2B (Conservation Genetics CAS Kun-ming Cell Bank, Chinese Academy Sciences), a widely used lung cell line, was cultured in BEGM™ bronchial epithelial cell growth medium (CC-3170, Lonza) and maintained in a humidified incubator with 5% CO₂ at 37 °C. For PM_2.5 exposure, each one-eighth filter with PM_2.5 samples was sonicated in 50 mL Milli-Q water for 30 min 5 times at 4 °C. The supernatants were freeze-dried and redissolved in 1 mL potassium phosphate buffer (PBS) (Thermo Fisher). Of each sample solution, 100 μL was diluted with growth media and added to BEAS-2B cells seeded in each well of 96-well plates. After 24 h exposure, cell viability and lactate dehydrogenase (LDH) release were detected by MTS assay (Promega, USA) and LDH cytotoxicity detection kit (Beyotime, Beijing) with six replicates (n = 6).

Song Y., Zhang Y., Zhu L., Chen Y., Chen Y.J., Zhu Z., Feng J., Qi Z., Yu J.Z., Yang Z, & Cai Z. (2024). Phosphocholine-induced energy source shift alleviates mitochondrial dysfunction in lung cells caused by geospecific PM2.5 components. Proceedings of the National Academy of Sciences of the United States of America, 121(14), e2317574121.

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Quantitative Metabolite Profiling Workflow

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Formic acid (FA), MS grade water, and acetonitrile (ACN) were obtained from Fisher Chemicals (Loughborough, UK). Adenosine-3′-phosphate 5′ phosphate lithium salt (PAPS), dimethyl sulfoxide, leucine enkephalin acetate salt, reduced L-glutathione (GSH), poly-DL-alanine, and sodium formate were purchased from Sigma-Aldrich (Buchs, Switzerland). Potassium phosphate buffer (0.5 M, pH 7.4) was provided by Thermo Fisher Scientific (Allschwil, Switzerland). Pooled liver S9 fractions were purchased from either BioIVT (West Sussex, UK) or Corning (Woburn, MA, USA). Reference material of investigated compounds was obtained either commercially from various vendors (SI, Table S1) or was synthesized in-house. Due to proprietary reasons, drug names, development codes, batch numbers, or obtained m/z values of utilized internal reference material of active development compounds cannot be disclosed. Corresponding material was denoted as either NVSx (parent drugs) or Mx (metabolites). Moreover, molecular structures of selected internal compounds can only be shown partially.

Lanshoeft C., Schütz R., Lozac’h F., Schlotterbeck G, & Walles M. (2023). Potential of measured relative shifts in collision cross section values for biotransformation studies. Analytical and Bioanalytical Chemistry, 416(2), 559-568.

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Detailed Biochemical Assay Protocols

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Chemicals were purchased from Sigma-Aldrich (Gillingham, Dorset, UK) unless otherwise specified. Homogenization buffer was phosphate-buffered saline (PBS) with 0.5 mM EDTA, 5 mM histidine, and 0.25 M sucrose, pH 7.4. Storage buffer was 100 mM Trizma with 0.5 mM EDTA in deionized water, pH 7.4. CYP assay buffer was 25 mM potassium phosphate buffer, pH 7.4, with 1.5% w/v potassium chloride and 30% v/v glycerol (Fisher Scientific, Loughborough, UK). G6Pase assay buffer was 100 mM BIS-TRIS, pH 6.5. Taussky-Shorr color reagent (Taussky and Shorr, 1953 (link)) was 0.18 M ferrous sulfate heptahydrate, 1% w/v ammonium molybdate in 0.5 M sulfuric acid.

Scotcher D., Billington S., Brown J., Jones C.R., Brown C.D., Rostami-Hodjegan A, & Galetin A. (2017). Microsomal and Cytosolic Scaling Factors in Dog and Human Kidney Cortex and Application for In Vitro-In Vivo Extrapolation of Renal Metabolic Clearance. Drug Metabolism and Disposition, 45(5), 556-568.

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Lipase-mediated PVC Endotracheal Tube Modification

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Sheridan^® PVC ETTs (Hudson RIC, Temecula, CA, USA) were modified using a fungal lipase produced from R. arrhizus (Sigma Aldrich, St Louis, MO, USA). PVC ETTs were cut into pieces with a length of 0.6 cm and a width of 0.3 cm. PVC pieces were then immersed in 20 mL of a 0.1% mass solution of R. arrhizus lipase dissolved in a 1M potassium phosphate buffer (Fisher Scientific, Waltham, MA, USA). These pieces were then gently agitated on an incubator shaker (Excella E24, New Brunswick Scientific, Edison, NJ, USA) for 24 hours at 37°C and 200 rpm. After 24 hours, the lipase solution was replaced and the ETT pieces were agitated for an additional 24 hours. At the end of 48 hours, the ETT pieces were removed from the lipase solution and were washed with distilled water. The ETT pieces were then sterilized using ethylene oxide exposure (Steri-Vac 5XL, 3M, Minneapolis, MN, USA) in a 16-hour sterilization cycle.
The activity of the R. arrhizus lipase used in this experiment was 10.5 U/g, where one unit is defined as the amount of enzyme that catalyzes the release of 1 μmol of oleic acid per minute at pH 7.4 and 40°C.20

Machado M.C, & Webster T.J. (2017). Lipase degradation of plasticized polyvinyl chloride endotracheal tube surfaces to create nanoscale features. International Journal of Nanomedicine, 12, 2109-2115.

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Antioxidant Activity Evaluation Chemicals

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2=2=-Azobis-(2-amidinopropane)-dihydrochloride (AAPH), fluorescein, DL-penicillamine, sodium hydroxide, potassium phosphate buffer, methanol, and acetone were purchased from Fisher Scientific (Waltham, Massachusetts, USA). Peroxynitrite was purchased from Calbiochem (Billerica, Massachusetts, USA). DHR123 (dihydrorhodamine 123), DTPA (diethylenetriaminepentaacetic acid), and all other chemicals were purchased from Sigma-Aldrich (St. Louis, Missouri, USA) and were of the highest purity available.

Babish J.G., Dahlberg C.J., Ou J.J., Keller W.J., Gao W., Kaadige M.R., Brabazon H., Lamb J., Soudah H.C., Kou X., Zhang Z., Pacioretty L.M, & Tripp M.L. (2016). Synergistic in vitro antioxidant activity and observational clinical trial of F105, a phytochemical formulation including Citrus bergamia, in subjects with moderate cardiometabolic risk factors. Canadian journal of physiology and pharmacology, 94(12).

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