Total Cholesterol Assay Kit (Colorimetric)
Contents
1. Product Manual Total Cholesterol Assay Kit Colorimetric Catalog Number STA 384 192 assays FOR RESEARCH USE ONLY Not for use in diagnostic procedures CELL BIOLABS INC Creating Solutions for Life Science Research Introduction Cholesterol is a lipid sterol that is produced in and transported throughout the bloodstream in eukaryotes Cholesterol is a critical compound used in the structure of cell membranes hormones and cell signaling It is an essential component of animal cell structure in order to maintain permeability and fluidity Cholesterol is a precursor for steroid hormones including the adrenal gland hormones cortisol and aldosterone sex hormones progesterone estrogens and testosterone and bile acids and vitamin D Cholesterol is transported throughout the body within lipoproteins which have cell specific signals that direct the lipids they transport to certain tissues For this reason lipoproteins exist in different forms within the blood based on their density These include chylomicrons very low density lipoproteins VLDLs low density lipoproteins LDLs intermediate density lipoproteins IDLs and high density lipoproteins HDLs The higher the lipid content within a lipoprotein the lower its density Cholesterol exists within a lipoprotein as a free alcohol and as a fatty cholesteryl ester which is the predominant form of cholesterol transport and storage Determining circulatory levels of lipoproteins i2. Lipid modulating effects of aqueous extract of Rubus occidentalis in hepatocarcinoma HepG2 cells American Journal of Bioscience 2 64 69 4 Pr v raud D P et al 2014 Effect of the type of dietary triacylglycerol fatty acids on a tocopherol concentration in plasma and tissues of growing pigs J Anim Sci 92 4972 4980 5 Raveendran W et al 2014 Hl antihistamines exacerbate high fat diet induced hepatic steatosis in wild type but not in apolipoprotein E knockout mice Am J Physiol Gastrointest Liver Physiol 307 G219 G228 6 Marino A et al 2014 ITCH deficiency protects from diet induced obesity Diabetes 63 550 561 7 Mathews E et al 2014 Mutation of 3 hydroxy 3 methylglutaryl CoA synthase I reveals requirements for isoprenoid and cholesterol synthesis in oligodendrocyte migration arrest axon wrapping and myelin gene expression J Neurosci 34 3402 3412 Warranty These products are warranted to perform as described in their labeling and in Cell Biolabs literature when used in accordance with their instructions THERE ARE NO WARRANTIES THAT EXTEND BEYOND THIS EXPRESSED WARRANTY AND CELL BIOLABS DISCLAIMS ANY IMPLIED WARRANTY OF MERCHANTABILITY OR WARRANTY OF FITNESS FOR PARTICULAR PURPOSE CELL BIOLABS s sole obligation and purchaser s exclusive remedy for breach of this warranty shall be at the option of CELL BIOLABS to repair or replace the products In no event shall CELL BIOLABS be liable for any proximate in
3. minimize possible interfering compounds Run proper controls as necessary Always run a standard curve with samples Tissue Lysates For 10 mg of tissue extract with 200 uL of a mixture of chloroform isopropanol NP 40 7 11 0 1 in a micro homogenizer Centrifuge the extract 10 minutes at 15 000 x g Transfer the liquid organic phase to a new tube taking care to avoid the pellet Air dry at 50 C to remove the chloroform Put samples under vacuum for 30 minutes to remove the trace amounts of organic solvent Dissolve the dried lipids in 200 uL of 1X Assay Diluent with sonicating and vortexing until the solution is homogenous the solution may appear cloudy This extraction procedure may be scaled up if larger sample amounts are desired Use 1 50 uL of extracted sample per assay Next adjust the volume to 50 uL per well with 1X Assay Diluent For unknown samples we suggest testing different amounts of samples to ensure that the readings are within the linear portion of the standard curve Cell Lysates Wash cells 3 times with cold PBS prior to lysis For 10 cells extract with 200 uL of a mixture of chloroform isopropanol NP 40 7 11 0 1 in a micro homogenizer Centrifuge the extract 10 minutes at 15 000 x g Transfer the liquid organic phase to a new tube taking care to avoid the pellet Air dry at 50 C to remove the chloroform Put samples under vacuum for 30 minutes to remove the trace amounts of organic solvent Dissolve the dri
4. ase Reconstitute the powder with 200 uL of 1X Assay Diluent Vortex vigorously until dissolved Prepare aliquots and store at 20 C to avoid multiple freeze thaws of the reconstituted powder e Cholesterol Reaction Reagent Prepare the reagent by diluting the Cholesterol Oxidase 1 50 HRP 1 50 Colorimetric Probe 1 50 and Cholesterol Esterase 1 250 in 1X Assay Diluent e g For 100 assays combine 100 uL of Cholesterol Oxidase 100 uL of HRP 100 uL Colorimetric Probe and 4 CELL BIOLABS INC A a 20 uL Cholesterol Esterase with 1X Assay Diluent to 5 mL total solution Mix thoroughly and protect the solution from light For best results place the Cholesterol Reaction Reagent on ice and use within 30 minutes of preparation Do not store the Cholesterol Reaction Reagent solution Notes 1 If testing for the concentration of free cholesterol is needed only omit the addition of Cholesterol Esterase from the Cholesterol Reaction Reagent solution 2 The Colorimetric Probe is light sensitive and must be stored accordingly Preparation of Samples Samples should be assayed immediately or stored at 80 C prior to performing the assay Optimal experimental conditions for samples must be determined by the investigator The following recommendations are only guidelines and may be altered to optimize or complement the user s experimental design A set of serial dilutions is recommended for samples to achieve optimal assay results and
5. cidental or consequential damages in connection with the products 8 CELL BIOLABS INC Contact Information Cell Biolabs Inc 7758 Arjons Drive San Diego CA 92126 Worldwide 1 858 271 6500 USA Toll Free 1 888 CBL 0505 E mail tech cellbiolabs com www cellbiolabs com 2012 2015 Cell Biolabs Inc All rights reserved No part of these works may be reproduced in any form without permissions in writing 9 CELL BIOLABS INC Wa SN
6. e 1 Cholesterol Standard Part No 239001 One 50 uL tube of a 10 mM cholesterol solution in ethanol 2 Assay Diluent 5X Part No 239002 One 100 mL bottle 50X Colorimetric Probe Part No 238401 One 200 uL tube in DMSO 4 HRP Part No 234402 Two 100 uL tubes of 100 U mL HRP solution in glycerol Box 2 shipped on blue ice packs 1 Cholesterol Esterase Part No 239003 One tube of 10 Units enzyme in powder 2 Cholesterol Oxidase Part No 239004 One 200 uL tube Materials Not Supplied 1 96 well microtiter plates Distilled or deionized water 1X PBS 10 uL to 1000 uL adjustable single channel micropipettes with disposable tips 50 uL to 300 uL adjustable multichannel micropipette with disposable tips Multichannel micropipette reservoir Spectrophotometric microplate reader capable of reading in the 540 570 nm absorbance range Oy oP YS Superoxide dismutase optional Storage Upon receipt store the Assay Diluent at 4 C Store the remaining kit components at 20 C The 50X Colorimetric Probe is light sensitive and must be stored accordingly Avoid multiple freeze thaw cycles Preparation of Reagents e 1X Assay Diluent Warm the Assay Diluent 5X to room temperature prior to using Dilute the Assay Diluent 5X with deionized water by diluting the 100 mL Diluent with 400 mL deionized water for 500 mL total Mix to homogeneity Store the 1X Assay Diluent at 4 C up to six months e Cholesterol Ester
7. ed lipids in 200 uL of 1X Assay Diluent with sonicating and vortexing until the solution is homogenous the solution may appear cloudy This extraction procedure may be scaled up if larger sample amounts are desired Use 1 50 uL of extracted sample per assay Next adjust the volume to 50 uL per well with 1X Assay Diluent For unknown samples we suggest testing different amounts of samples to ensure that the readings are within the linear portion of the standard curve Serum Collect blood in a tube with no anticoagulant Allow the blood to clot at room temperature for 30 minutes Centrifuge at 2500 x g for 20 minutes Remove the serum layer and store on ice Avoid disturbing the white buffy layer Aliquot samples for testing and store at 80 C Perform dilutions in 1X Assay Diluent Serum samples must be diluted at least 1 100 to 1 200 with Assay Diluent This will provide values within the range of the standard curve Cholesterol levels in serum average about 3 higher in value than in the corresponding plasma pair Ref 2 Plasma Avoid hemolyzed and lipemic blood samples Collect blood with heparin or citrate and centrifuge at 2000 x g and 4 C for 10 minutes Remove the plasma layer and store on ice Avoid disturbing the white buffy layer Aliquot samples for testing and store at 80 C Perform dilutions 5 h CELL BIOLABS INC yA n in 1X Assay Diluent Plasma samples must be diluted at least 1 100 to 1 200 with Assay Diluent Th
8. is will provide values within the range of the standard curve Notes 1 Samples with NADH concentrations above 10 uM and glutathione concentrations above 50 uM will oxidize the probe and could result in erroneous readings To minimize this interference it is recommended that superoxide dismutase SOD be added to the reaction at a final concentration of 40 U mL 2 Avoid samples containing DTT or mercaptoethanol since the colorimetric probe is not stable in the presence of thiols above 10 uM Preparation of Cholesterol Standard Curve 1 Prepare fresh cholesterol standards before use by diluting in 1X Assay Diluent First dilute the stock Cholesterol Standard 10 mM solution 1 40 in 1X Assay Diluent for a 250 uM solution eg add 25 uL of the stock 10 mM standard to 975 uL of 1X Assay Diluent Vortex thoroughly Use the diluted Cholesterol Standards promptly Use this 250 uM solution to prepare a series of the remaining cholesterol standards according to Table 1 below 250 uM Cholesterol 1X Assay Diluent Resulting Cholesterol Tubes Standard uL uL Concentration uM 1 1000 0 250 2 500 of Tube 1 500 125 3 500 of Tube 2 500 62 5 4 500 of Tube 3 500 31 3 5 500 of Tube 4 500 15 6 6 500 of Tube 5 500 7 8 7 500 of Tube 6 500 3 9 8 500 of Tube 7 500 1 9 9 500 of Tube 8 500 1 0 10 0 1000 0 Table 1 Preparation of Cholesterol Standards Note Do not store diluted chole
9. n of the cholesterol standards from Table 1 to determine the best curve See Figure 2 for an example standard curve 3 Determine the cholesterol concentration of the samples with the equation obtained from the linear regression analysis of the standard curve Substitute the corrected absorbance values for each sample Remember to account for dilution factors Sample corrected absorbance Total Cholesterol uM _______LLYC x Sample dilution Slope Cholesteryl Ester uM Total Cholesterol Free Cholesterol 7 N CELL BIOLABS INC l Note For the conversion of results from uM to mg dl divide the cholesterol concentration uM by 25 9 References 1 Admundson D M et al 1999 J Biochem Biophys Meth 38 43 52 2 Cholesterol and Triglyceride concentrations in Serum Plasma Pairs 1977 Clin Chem 23 60 63 3 Fossati P et al 1982 Clin Chem 28 2077 2080 4 Ledwozyw A et al 1986 Clin Chim Acta 155 275 284 5 Lee S M et al 2008 Lipids 43 419 429 Recent Product Citations 1 Parisi O I et al 2015 Sericin poly ethylcyanoacrylate nanospheres by interfacial polymerization for enhanced bioefficacy of fenofibrate in vitro and in vivo studies Biomacromolecules doi 10 1021 acs biomac 5b00746 2 Mignarri A et al 2015 Evaluation of cholesterol metabolism in cerebrotendinous xanthomatosis J Inherit Metab Dis doi 10 1007 s10545 015 9873 1 3 Moon Y S et al 2014
10. s critical to the diagnosis of lipid transport disorders High levels of cholesterol and cholesteryl esters hypercholesterolemia have been associated with cardiovascular disease such as atherosclerosis and heart disease although lower levels hypocholesterolemia may be associated with cancer depression or respiratory diseases Cell Biolabs Total Cholesterol Assay Kit is a simple colorimetric assay that measures the amount of total cholesterol present in plasma serum tissue homogenates or cell lysates in a 96 well microtiter plate format The assay will detect total cholesterol cholesteryl esters plus free cholesterol in the presence of cholesterol esterase or only free cholesterol in the absence of the esterase enzyme Each kit provides sufficient reagents to perform up to 192 assays including blanks cholesterol standards and unknown samples Sample cholesterol concentrations are determined by comparison with a known cholesterol standard Cholesteryl esters can be quantified by subtracting the free cholesterol values from the total cholesterol value Assay Principle Cell Biolabs Total Cholesterol Assay Kit measures the total cholesterol within serum plasma lysate or tissue samples The assay is based on the enzyme driven reaction that quantifies both cholesterol esters and free cholesterol Cholesterol esters are hydrolyzed via cholesterol esterase into cholesterol which is then oxidized by cholesterol oxidase into the ketone chole
11. st 4 en 3 one plus hydrogen peroxide The hydrogen peroxide is then detected with a highly specific colorimetric probe Horseradish peroxidase catalyzes the reaction between the probe and hydrogen peroxide which bind in a 1 1 ratio Samples are compared to a known concentration of cholesterol standard in a 96 well microtiter plate format Samples and standards are incubated for 45 minutes and then read with a standard 96 well colorimetric plate reader Figure 1 2 les CELL BIOLABS INC A a gsr Cholesteryl Ester Cholestery Oleate Cholesterol Esterase Cholesterol Cholesterol Oxidase Colorimetric Probe 2 2 HRP OD H O 540 570 nm Figure 1 Colorimetric Cholesterol Assay Principle Related Products 1 Oo OS OY OS e ee RW N e O STA 241 STA 242 STA 243 STA 361 STA 362 STA 363 STA 364 STA 365 STA 366 STA 367 STA 368 STA 369 STA 390 STA 391 Human Low Density Lipoprotein Human Very Low Density Lipoprotein Human High Density Lipoprotein Human ApoAI and ApoB Duplex ELISA Kit Human ApoAI ELISA Kit Human ApoAII ELISA Kit Human ApoCI ELISA Kit Human ApoCII ELISA Kit Human ApoCII ELISA Kit Human ApoE ELISA Kit Human ApoB 100 ELISA Kit OxiSelect Human Oxidized LDL ELISA Kit MDA LDL Quantitation Total Cholesterol Assay Kit Fluorometric HDL and LDL VLDL Cholesterol Assay Kit i N CELL BIOLABS INC JA SA N Kit Components Box 1 shipped at room temperatur
12. sterol standard solutions Assay Protocol Each cholesterol standard and sample should be assayed in duplicate or triplicate A freshly prepared standard curve should be used each time the assay is performed l 2 Add 50 uL of the diluted cholesterol standards or samples to a 96 well microtiter plate Add 50 uL of the prepared Cholesterol Reaction Reagent to each well and mix the well contents thoroughly Cover the plate wells to protect the reaction from light Incubate the plate for 45 minutes at 37 C AN 5 CELL BIOLABS INC JE a 4 IMMEDIATELY read the plate with a spectrophotometric microplate reader in the 540 570 nm range 5 Calculate the concentration of cholesterol within samples by comparing the sample absorbance values to the cholesterol standard curve Example of Results The following figures demonstrate typical Total Cholesterol Assay results One should use the data below for reference only This data should not be used to interpret or calculate actual sample results a z q iN Q O v Z 100 150 200 Cholesterol uM Figure 2 Cholesterol Standard Curve Calculation of Results 1 Calculate the average absorbance values for every standard control and sample Subtract the average zero standard value from itself and all standard and sample values This is the corrected absorbance 2 Plot the corrected absorbance for the standards against the final concentratio
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