Diacerein_13739-02-1_DataSheet_MedChemExpress

Endotoxin Removal Solution Catalog Number E4274Product DescriptionEndotoxins are lipopolysaccharides (LPS), a major component of the Gram-negative bacterial cell wall, and are commonly found as contaminants in plasmid DNA preparations from E. coli. Endotoxins are large, negatively charged molecules that co-purify with DNA on ion exchange and size exclusion columns and in CsCl banding. Endotoxins are extremely potent stimulators of the mammalian immune system and are toxic to primary cells and to animals. The endotoxin toxicity is an obstacle to in vitro and in vivo transfection experiments.Non-ionic detergents, traditionally used for separation of integral membrane proteins,1 can be utilized for removal of endotoxins from DNA solutions by phase separation.2The solubility behavior of a detergent in a dilute, aqueous solution at physiological salt and pH conditions is strongly dependent upon the temperature of the solution. At low temperatures, the detergent forms a clear, micellar solution, but above the cloud point temperature, the micelles form larger, turbid aggregates and ultimately fuse to form a separate phase. The lower phase is detergent-enriched and the detergent-depleted upper phase contains detergent at a concentration slightly above the critical micellar concentration (CMC). Amphiphilic and hydrophobic molecules associated with the micelles of the detergent will aggregate within the detergent-enriched phase, while the soluble, hydrophilic molecules will remain in the detergent-depleted upper phase.Extraction of endotoxin contaminated DNA solutions with the appropriate non-ionic detergent will separate the hydrophilic DNA from the amphiphilic endotoxin. The amphiphilic endotoxin will associate with the lower phase, while the DNA will remain in the upper, detergent-depleted phase.2Reagents and equipment required, but not provided • Water, Molecular Biology Reagent, Catalog Number W4502• E-TOXATE® Water, Catalog Number 2107, or Tris-EDTA (TE) buffer 100×, Catalog NumberT9285• DNA solution (0.5 ml), ~ 1 mg/ml in E-TOXATE®Water or TE buffer• 3 M sodium acetate solution, pH 7.5.• 2-Propanol, Catalog Number I9516, or Ethanol, 190 proof, Catalog Number E7148; 200 proof, CatalogNumber E7023• 70% Ethanol• E-TOXATE®reagents Kits, Catalog Numbers 210A1, 210B1 or 210C1• Ice bucket• Heat block or incubator at 37 °C• Microcentrifuge at room temperature• 1.5 or 2 ml sterile microcentrifuge tubes• Endotoxin-free pipet tips (40-200 µl, 200-1000 µl) Precautions and DisclaimerThis product is for R&D use only, not for drug, household, or other uses. Please consult the Material Safety Data Sheet for information regarding hazards and safe handling practices.StorageStore at room temperature.Note: Removal of endotoxins from DNA preparations can be performed either during the final stage of DNApreparation, or during an earlier stage.Procedures for Endotoxin RemovalDuring the final stage of DNA preparationNote: The procedure described below was performed on plasmid DNA produced in E. coli DH5α cells.• Losses of up to 50% of the DNA are expected. • Use of a DNA concentration above therecommended 1 mg/ml reduces the efficiency ofthe procedure.1. Pipette 500 µl of the DNA solution into a sterilemicrocentrifuge tube.2. Add 50 µl of the 3 M sodium acetate solution to theDNA sample.3. Incubate on ice for 5 minutes.4. Add 100 µl of cold Endotoxin Removal Solution.5. Mix thoroughly and incubate on ice for 10 minutes.The solution should be light blue and clear.6. Incubate the tube at 37 °C for 20 to 30 minutes oruntil the phases separate.7. Spin for 5 minutes at 3000 x g in themicrocentrifuge. The upper phase is colorless and clear, while the lower phase is blue.8. Carefully transfer the upper phase containing theDNA to a clean microcentrifuge tube.9. Repeat steps 4 through 8 twice.10. Add 0.6× volume of 2-propanol. Mix by inversion atroom temperature and centrifuge at 15,000 x g for30 minutes at 4 °C. Alternatively, add2.5× volumes of ethanol. Incubate overnight at –20°C or 20 minutes at –70 °C and centrifuge at15,000 x g for 30 minutes at 4 °C.11. Carefully remove the supernatant12. Wash the DNA pellet twice with cold 70% ethanol.Remove the supernatant.13. Air-dry the pellet.14. Suspend the DNA in 100 µl of endotoxin free wateror TE buffer.15. Determine DNA concentration and endotoxin levelsusing endotoxin assay reagents and compare tothe starting material. During an earlier stage of DNA preparationThis procedure is based on the alkaline lysis of E. coli DH5α cells.3 The endotoxins are removed immediately after alkaline cell lysis, neutralization, and a clarification step. The resulting high salt solution is suitable for the endotoxin removal step. It is performed under “endotoxin free” conditions. The plasticware used is either sterile and disposable, or NaOH-treated. The buffers are prepared with endotoxin free water.1. Add the Endotoxin Removal Solution (0.2× volume)to the cold, crude DNA solution.2. Incubate on ice and mix occasionally by inversionto obtain a homogenous, clear blue solution3. Incubate at 37 °C for 20 to 30 minutes until thephase separation is obvious.4. Spin for 5 minutes at low speed (3000 x g) at roomtemperature.5. Transfer the upper aqueous phase to an endotoxinfree container.6. Proceed with the DNA purification by any method.Use endotoxin-free buffers and containers. References1. Bordier, C., J. Biol. Chem., 256, 1604-1607, (1981).2. Cotten, M. et al., Gene Therapy, 1, 239-246,(1994).3. Sambrook et al., Molecular Cloning, a LaboratoryManual, 2nd Ed. p. 1.38RK,PHC 09/05-1Sigma brand products are sold through Sigma-Aldrich, Inc.Sigma-Aldrich, Inc. warrants that its products conform to the information contained in this and other Sigma-Aldrich publications. Purchaser must determine the suitability of the product(s) for their particular use. Additional terms and conditions may apply. Please see reverse side ofthe invoice or packing slip.。

Product Information MINIMUM ESSENTIAL MEDIUM EAGLED-VALINE MODIFICATIONProduct Number M7395Storage Temperature 2-8°CProduct DescriptionMinimum Essential Medium (MEM), developed by Harry Eagle, is one of the most widely used of all synthetic cell culture media. Early attempts to cultivate normal mammalian fibroblasts and certain subtypes of HeLa cells revealed that they had specific nutritional requirements that could not be met by Eagle’s Basal Medium (BME). Subsequent studies using these and other cells in culture indicated that additions to BME could be made to aid growth of a wider variety of fastidious cells. MEM, which incorporates these modifications, includes higher concentrations of amino acids. MEM has been used for cultivation of a wide variety of cells grown in monolayers. Optional supplementation of non-essential amino acids to the formulations that incorporate either Hanks’ or Earle’s salts has broadened the usefulness of this medium. The formulation has been further modified by optional elimination of calcium to permit growth of cells in suspension culture.MINIMUM ESSENTIAL MEDIUM EAGLE, Product No. M 7395 is one of the cell culture media available from Sigma. The selection of a nutrient medium is strongly influenced by 1] type of cell, 2] type of culture [monolayer, suspension, clonal] and 3] degree of chemical definition necessary. It is important to review the literature for recommendations concerning medium, supplementation and physiological parameters required for a specific cell line.Components g/L Calcium Chloride•2H2O0.265 Magnesium Sulfate (anhydrous)0.09767 Potassium Chloride0.4 Sodium Chloride 6.8 Sodium Phosphate Monobasic0.122 (anhydrous)L-Arginine•HCl0.126L-Cystine•2HCl0.0313 L-Glutamine0.292L-Histidine•HCl•H2O0.042L-Isoleucine0.052L-Leucine0.052L-Lysine•HCl0.0725 L-Methionine0.015L-Phenylalanine0.032L-Threonine0.048L-Tryptophan0.01L-Tyrosine•2Na•2H2O0.0519D-Valine0.092 Choline Chloride0.001Folic Acid0.001myo-Inositol0.002 Niacinamide0.001D-Pantothenic Acid (hemicalcium)0.001 Pyridoxal•HCl0.001 Riboflavin0.0001 Thiamine•HCl0.001 Glucose 1.0 Phenol Red•Na0.011Precautions and DisclaimerREAGENTFor In Vitro Diagnostic UsePreparation InstructionsPowdered media are extremely hygroscopic and should be protected from atmospheric moisture. The entire contents of each package should be used immediately after opening. Preparing a concentrated solution of medium is not recommended as precipitates may form. Supplements can be added prior to filtration or introduced aseptically to sterile medium. The nature of the supplement may affect storage conditions and shelf life of the medium.1.Measure out 90% of final required volume ofwater. Water temperature should be 15-20°C.2.While gently stirring the water, add thepowdered medium. Stir until dissolved. Do NOTheat.3.Rinse original package with a small amount ofwater to remove all traces of powder. Add tosolution in step 2.4.To the solution in step 3, add 2.2 g sodiumbicarbonate or 29.3 ml of sodium bicarbonatesolution [7.5%w/v] for each liter of final volumeof medium being prepared. Stir until dissolved.5.While stirring, adjust the pH of the medium to0.1-0.3 pH units below the desired pH since itmay rise during filtration. The use of 1N HCl or1N NaOH is recommended.6.Add additional water to bring the solution tofinal volume.7.Sterilize immediately by filtration using amembrane with a porosity of 0.22 microns.8.Aseptically dispense medium into sterilecontainer.Storage/StabilityStore the dry powdered medium at 2-8°C under dry conditions and liquid medium at 2-8°C in the dark. Deterioration of the powdered medium may be recognized by any or all of the following: [1] color change, [2] granulation/clumping, [3] insolubility. Deterioration of the liquid medium may be recognized by any or all of the following: [1] pH change, [2] precipitate or particulate matter throughout the solution, [3] cloudy appearance [4] color change. The nature of supplements added may affect storage conditions and shelf life of the medium. Product label bears expiration date.ProcedureWater for tissue culture use [W-3500]Sodium Bicarbonate [S-5761] orSodium Bicarbonate Solution, 7.5% [S-8761]1N Hydrochloric Acid [H-9892]1N Sodium Hydroxide [S-2770]Medium additives as requiredProduct ProfileAppearance off-white powder Moisture content 2.0% Solubility clear solution at 1x concentrationpH at room temperature 5.8 ± 0.3 [without sodium bicarbonate]pH at room temperature 7.5 ± 0.3 [with sodium bicarbonate]Osmolality250 mOsm/kg H2O ± 5% [without sodium bicarbonate]Osmolality290 mOsm/kg H2O ± 5% [with sodium bicarbonate]Amino Acid Analysis Analysis has confirmedby HPLC that amino acids are present atconcentrations consistent withthe formula.Key Element Analysis Analysis has confirmed that by ICAP key elements are present atconcentrations consistent withthe formula.BIOLOGICAL PERFORMANCE CHARACTERISTICS Biological performance is assessed using an appropriate cell line(s). Growth studies are carried through 2 subculture generations. Cells are counted and growth is plotted as a logarithmic function of time in culture. Seeding efficiencies, doubling time, and final cell densities are determined. During the testing period cultures are examined microscopically for atypical morphology and evidence of cytotoxicity. Test results are available upon request.References1.Eagle, H. et al (1956) myo-Inositol as anEssential Growth Factor for Normal andMalignant Human Cells in Tissue Culture.J.Biol. Chem. 214, 845-847.2.Eagle, H.(1976) Media for Animal Cell Culture.Tissue Culture Association Manual. 3, 517-520.3.Eagle, H. (1959). Amino Acid Metabolism inMammalian Cell Cultures. Science. 130, 432-437.4.Eagle, H. (1955) Nutrition Needs of MammalianCells in Culture. Science. 122, 501.5.Gilbert, S.F. and Migeon, B.R. (1975) D-valineas a selective agent for normal human androdent epithelial cells in culture. Cell. 5, 11-17.7H027Sigma brand products are sold through Sigma-Aldrich, Inc.Sigma-Aldrich, Inc. warrants that its products conform to the information contained in this and other Sigma-Aldrich publications. Purchaser must determine the suitability of the product(s) for their particular use. Additional terms and conditions may apply. Please see reverse side ofthe invoice or packing slip.。

Manual ProcedureAutomated procedure on requestTest PrincipleFerric ions (Fe +3) are released from transferring under acidicconditions and reduced to ferrous state (Fe +2) by a strong reducing agent. Ferrous ions then react with Ferrozine to form a coloredcomplex which can be measured photometrically. The intensity of the color produced is proportional to the iron concentration in the sample. Lipemic samples are clarified by the detergent.Transferrin-Fe-complex apotransferrin + Fe 3+Fe 3+ Fe 2+Ferrozine + Fe 2+ colored complexStability and preparation of working reagentBuffer Reagent B-R1: liquid Powder Reagent P-R1: powder. Reagent R2: liquid, ready to useWorking ReagentAdd 10 ml reagent Buffer B-R1 to one vial powder reagent P-R1 andmix gently for 15 minutes before use.Stability: 2 weeks at 20 – 25 °C1 month at2 – 8 °CAll reagents are stable up to expiry date given on label when storedat 2 – 8 °CNote : All reagents should be clear solutions. Don’t use if the reagent is turbid. Specimen Collection and Handling1. Non-hemolyzed serum is the specimen of choice.2. Serum should be separated from blood clot as soon as possible.3. Heparinized plasma could only be used, other anticoagulantsshould not be used.4. Serum iron is reported to be stable for 4 days at 20 – 25°C and7 days at 2 – 8 °C.CalibratorMediCal U Cat. No. 15011Iron STD. Cat. No. 16131Quality controlMeditrol N Cat. No. 15171 Meditrol P Cat. No. 15181Iron (Ferrozine)Colorimetric testPowder liquid reagentsCalculationConc.Iron = ⨯ Conc. Standardμμ g /dlLinearity Up to 1000 μg/dl (179 μmol/L); If result exceeds 1000 μg/dl, repeat test using diluted sample (1+1) with sodium chloride solution (0.9 %) and multiply result by 2.Interference 1. Certain drugs and other substances are known to influencecirculating iron levels. 2. To make tubes, pipettes, etc. iron free, they must be washed with diluted (1+2) hydrochloric or nitric acid followed by several rinsings with iron free deionized or distilled water.Precautions 1. The reagent is toxi c, don’t pipette by mouth, avoid all contacts. 2. Use only disposable plastic containers or iron free tubes andcuvettes. Avoid any contamination by the use of clean laboratory material.Cat. No. 12881 B-R1 1 Buffer 50mlFor 50 testsP-R1 5 powder for 10 mlR2 1 x 10 ml(A S -A SB ) SampleA standardGuanidinium PH=4.5 Ascorbic acidIron ferrozine methodReference rangeReferences1. Garoc A., Clin. Chem. Acta 94, 115 (1979).2. Brivio et coll., La Ricerca Clin. Lab. 18, 523 (1986).3. Young, DS., Effects of Drugs on Clinical Laboratory Tests, fifthedition 2000, AACC Press, Washington, D.C.。

收稿日期：2021-08-12；修订日期：2021-11-16作者信息：杜秀明，E-mail：**********。

*周庆云，E-mail：**********采用体外染色体畸变试验检测锐钛型纳米二氧化钛的遗传毒性杜秀明1，2，洪丽玲1，2，徐灵芝1，2，周庆云1，2，* (1．上海化工研究院有限公司，上海200062；2．上海化工院检测有限公司，上海200062)In vitro chromosome aberrationevaluation of anatase titaniumdioxide nanoparticlesDU Xiuming1,2,HONG Liling1,2,XU Lingzhi1,2,ZHOU Qingyun1,2,*(1.Testing Center,Shanghai Research Institute of Chemical IndustryCo.,Ltd.,Shanghai200062；2.Shanghai Institute of ChemicalIndustry Testing Co.,Ltd.,Shanghai200062,China)【摘要】目的：按《OECD化学品测试准则473：体外哺乳动物染色体畸变测试》的要求进行试验，检测锐钛型纳米二氧化钛诱发体外培养的哺乳动物细胞染色体畸变的能力，以评价其是否属于致突变物。

方法：试验组受试物终浓度分别设置为0.0078、0.0312、0.125、0.5和2mg/mL，同时设立溶剂对照组和阳性对照组。

试验分为有代谢活化系统短时处理组(+S9，4h)、无代谢活化系统短时处理组(-S9，4h)和无代谢活化系统连续处理组(-S9，24h)3种处理方式，将培养的中国仓鼠肺细胞(CHL)暴露于锐钛型纳米二氧化钛混悬液中，染毒相应时间后收获细胞，经低渗固定后，制片并染色。

每个浓度组选取300个分散良好的中期分裂相细胞进行染色体畸变分析。