Total Cholesterol Assay Kit (Fluorometric)
Contents
1. Product Manual Total Cholesterol Assay Kit Fluorometric Catalog Number STA 390 192 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. for the standards against the final concentration 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 fluorescence values for each sample Remember to account for dilution factors Sample corrected fluorescence Total Cholesterol uM _ __ x Sample dilution Slope Cholesteryl Ester uM Total Cholesterol Free Cholesterol 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 g IN CELL BIOLABS INC JA Ea N 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 Joseph B K et al 2015 Inhibition of AMP kinase by the protein phosphatase 2A heterotrimer PP2APpp2r2d J Biol Chem doi 10 1074 jbc M114 626259 2 Rodriguez Jorquera I A et al 2015 Transcriptional and physiological response of fathead minnows Pimephales promelas exposed to urban waters entering into wildlife protected areas Environ
3. 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 i CELL BIOLABS INC om 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 1 200 to 1 400 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 in 1X Assay Diluent Plasma samples must be diluted at least
4. 1 200 to 1 400 with Assay Diluent This 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 B mercaptoethanol since the fluorescence probe is not stable in the presence of thiols above 10 uM Preparation of Cholesterol Standard Curve 1 Prepare fresh cholesterol standards by diluting in 1X Assay Diluent First dilute the stock Cholesterol Standard 10 mM solution 1 50 in 1X Assay Diluent for a 200 uM solution eg add 20 uL of the stock 10 mM standard to 980 uL of 1X Assay Diluent Vortex thoroughly 2 Use this 200 uM solution to prepare a series of the remaining cholesterol standards according to Table 1 below 200 uM Cholesterol 1X Assay Diluent Resulting Cholesterol Tubes Standard uL uL Concentration uM 1 60 940 12 2 50 950 10 3 40 960 8 4 30 970 6 5 20 980 4 6 10 990 2 7 5 995 1 8 0 1000 0 Table 1 Preparation of Cholesterol Standards Note Do not store diluted cholesterol standard solutions Assay Protocol Each cholesterol standard and sample should be assayed in duplicate or triplicate A freshly prepared standard cu
5. Pollut 199 155 165 3 Liu Z H et al 2014 Abnormal lipid metabolism down regulates adenosine triphosphate synthase B subunit protein expression in corpus cavernosum smooth muscle in vitro and in vivo Andrologia 46 487 494 4 Ananth S et al 2014 Regulation of the cholesterol efflux transporters ABCA1 and ABCGI in retina in hemochromatosis and by the endogenous siderophore 2 5 dihydroxybenzoic acid Biochim Biophys Acta 1842 603 612 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 incidental or consequential damages in connection with the products 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 CELL BIOLABS INC
6. f serial dilutions is recommended for samples to achieve optimal assay results and 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 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 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
7. k plates Cholesterol Standard Part No 239001 One 50 uL tube of a 10 mM cholesterol solution in ethanol 3 Assay Diluent 5X Part No 239002 One 100 mL bottle Fluorescence Probe Part No 239005 One 200 uL tube in DMSO 5 HRP Part No 234402 Two 100 uL tubes of 100 U mL HRP solution in glycerol Box 2 shipped on blue ice packs 1 2 Cholesterol Esterase Part No 239003 One tube of 10 Units enzyme in powder Cholesterol Oxidase Part No 239004 One 200 uL tube Materials Not Supplied 1 Noe ees 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 Fluorescence microplate reader capable of reading excitation in the 530 570 nm range and emission in the 590 600 nm range Superoxide dismutase optional Storage Upon receipt store the Cholesterol Standard Fluorescence Probe HRP Cholesterol Oxidase and Cholesterol Esterase at 20 C The Fluorescence Probe is light sensitive and must be stored accordingly Avoid multiple freeze thaw cycles Store the remaining kit components at 4 C AN CELL BIOLABS INC JE D Preparation of Reagents 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
8. mL deionized water for 500 mL total Mix to homogeneity Store the 1X Assay Diluent at 4 C up to six months Cholesterol Esterase 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 Cholesterol Reaction Reagent Prepare the reagent by diluting the Cholesterol Oxidase 1 50 HRP 1 50 Fluorescence Probe 1 50 and Cholesterol Esterase 1 250 in 1X Assay Diluent eg For 100 assays combine 100 uL of Cholesterol Oxidase 100 uL of HRP 100 uL Fluorescence Probe and 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 Fluorescence 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 o
9. rve should be used each time the assay is performed 6 AN CELL BIOLABS INC JE D 1 Add 50 uL of the diluted cholesterol standards or samples to the 96 well microtiter plate 2 Add 50 uL of the prepared Cholesterol Reaction Reagent to each well and mix the well contents thoroughly 3 Cover the plate wells to protect the reaction from light Incubate the plate for 45 minutes at 37 C 4 IMMEDIATELY read the plate with a fluorescence microplate reader equipped for excitation in the 530 570 nm range and for emission in the 590 600 nm range 5 Calculate the concentration of cholesterol within samples by comparing the sample RFU 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 8 Cholesterol uM Figure 2 Cholesterol Standard Curve i N CELL BIOLABS INC gt LL 1500 p Vo z 0 5 10 15 20 25 30 35 Cholesterol Oleate pM Figure 3 Cholesterol Oleate tested in Total Cholesterol Assay Kit Calculation of Results 1 Calculate the average fluorescence 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 fluorescence 2 Plot the corrected fluorescence
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 fluorometric 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 fluorescence 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 within the 96 well microtiter plate format Samples and standards are incubated for 45 minutes and then read with a standard 96 well fluorometric plate reader Figure 1 2 les CELL BIOLABS INC A a r e Cholesteryl Ester Cholesteryl Oleate Cholesterol Esterase Cholesterol Cholesterol Oxidase AN H O Cholest 4 ene 3 one as Fluorescence Probe Figure 1 Cholesterol Assay Principle Related Products 1 STA 241 Human Low Density Lipoprotein STA 242 Human Very Low Density Lipoprotein STA 243 Human High Density Lipoprotein STA 361 Human ApoAI and ApoB Duplex ELISA Kit STA 362 Human ApoAI ELISA Kit STA 363 Human ApoAII ELISA Kit STA 364 Human ApoCI ELISA Kit Sl Go ee a 3 oe BIOLABS INC 8 2 STA 365 Human ApoCII ELISA Kit STA 366 Human ApoCII ELISA Kit 10 STA 367 Human ApoE ELISA Kit 11 STA 368 Human ApoB 100 ELISA Kit 12 STA 369 OxiSelect Human Oxidized LDL ELISA Kit MDA LDL Quantitation 13 STA 391 HDL and LDL VLDL Cholesterol Assay Kit Kit Components Box 1 shipped at room temperature 1 2 96 well Microtiter Plate Part No 234501 Two 96 well clear bottom blac
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