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Glycolysis Cell-Based Assay Kit Item No. 600450 Customer Service 800.364.9897 * Technical Support 888.526.5351 www.caymanchem.com TABLE OF CONTENTS GENERAL INFORMATION 3 Materials Supplied 4Precautions 4 If You Have Problems 4 Storage and Stability 4 Materials Needed but Not Supplied INTRODUCTION 5Background PRE-ASSAY PREPARATION ASSAY PROTOCOL Materials Supplied Kit will arrive packaged as a -20°C kit. For best results, remove components and store as stated below. Item Number Item Quantity/Size Storage 6 About This Assay 600451 Glycolysis Assay Substrate 1 vial/250 µl -20°C 7 Cell Culture Preparation 600452 Glycolysis Assay Enzyme Mixture 2 vials/lyophilized -20°C 600453 Glycolysis Assay L-Lactate Standard 1 vial/120 µl 4°C 600454 Glycolysis Assay Cofactor 1 vial/250 µl 4°C Cell-Based Assay Buffer Tablet 1 tablet Room Temperature 8 Reagent Preparation GENERAL INFORMATION 9 Standard Preparation 10 Plate Set Up 11 Performing the Assay P ERFORMANCE CHARACTERISTICS 12Calculations 12 Performance Characteristics RESOURCES 15Troubleshooting 16References 17 Related Products 10009322 If any of the items listed above are damaged or missing, please contact our Customer Service department at (800) 364-9897 or (734) 971-3335. We cannot accept any returns without prior authorization. ! WARNING: This product is for laboratory research use only: not for administration to humans. Not for human or veterinary diagnostic or therapeutic use. 18 Warranty and Limitation of Remedy 19 Plate Template 20Notes GENERAL INFORMATION 3 Precautions Please read these instructions carefully before beginning this assay. For research use only. Not for human or diagnostic use. If You Have Problems Technical Service Contact Information Phone: 888-526-5351 (USA and Canada only) or 734-975-3888 Fax:734-971-3641 Email:[email protected] Hours: M-F 8:00 AM to 5:30 PM EST In order for our staff to assist you quickly and efficiently, please be ready to supply the lot number of the kit (found on the outside of the box). Storage and Stability This kit will perform as specified if stored at -20°C and used before the expiration date indicated on the outside of the box. Materials Needed But Not Supplied 1. A plate reader with the capability to measure absorbance between 490-520 nm 2. A 96-well plate for culturing cells 3. A plate centrifuge 4. Adjustable pipettes and a repeating pipettor 5. A source of pure water; glass distilled water or HPLC-grade water is acceptable 4 GENERAL INFORMATION INTRODUCTION Background Glycolysis is one of the major metabolic pathways in glucose metabolism. It is a process that is conserved throughout evolution and occurs in virtually all cells. Glycolysis is the primary source of ATP when oxygen is limiting, such as during intense strenuous exercise and also in erythrocytes which lack mitochondria. The balance between gluconeogenesis and glycolysis in responding to the needs of the entire body is tightly regulated by intracellular molecules such as amino acids, and external factors such as insulin, which stimulates glycolysis. In humans, various types of cells display different glycolytic pathways. For example, neurons depend absolutely on aerobic glycolysis for energy whereas muscles derive almost all ATP from anaerobic glycolysis.1 Over 80 years ago, Otto Warburg observed that cancer cells preferentially obtained energy from aerobic glycolysis, which produces significantly higher amounts of lactate from pyruvate, whereas normal cells use the aerobic tricarboxylic acid cycle, which oxidizes pyruvate to carbon dioxide and water. In fact, the conversion of glucose to lactate in the presence of oxygen is a critical aerobic pathway that allows cancer cells to proliferate rapidly and the amount of lactate produced is correlated with tumor aggressiveness.2,3 It is known that both embryonic stem cells and cancer cells share common metabolic profiles with elevated glycolytic activity that might contribute to the proliferative potential of these cells. Furthermore, the increase in glycolysis is partially mediated by hypoxia-inducible transcriptional factor (HIF-1). However, the mechanism behind this metabolic shift is not well understood.4 Lactate is the end product of glycolysis and is released into the extracellular environment. Extracellular lactate levels are proportionally correlated with intracellular glycolytic activity and thus have been used as metabolic biomarkers of tumors.2 In cancer research, direct inhibiton of glycolysis has long been a chemotherapeutic strategy.5 Measuring lactate levels is part of metabolomic approaches in drug discovery to monitor the effects of anticancer drugs. INTRODUCTION 5 About This Assay Cayman’s Glycolysis Cell-Based Assay Kit provides a colorimetric-based method for detecting L-lactate in culture medium. It can also be utilized to determine intracellular lactate concentrations in cultured cells. In the assay, lactate dehydrogenase catalyzes the oxidation of lactate to pyruvate, in which the formed NADH reduces a tetrazolium substrate (INT) to a highly-colored formazan which absorbs strongly at 490-520 nm. The amount of formazan produced is proportional to the amount of lactate released into the culture medium and can be used as an indicator of cellular glycolytic rate. This single step, no-wash assay can be adapted to high-throughput screening for glycolysis regulators at the cellular level. Furthermore, the non-invasive sampling leaves the cells intact and thus allows multiplex testing for additional markers. 6 INTRODUCTION PRE-ASSAY PREPARATION Cell Culture Preparation 1. Seed cells in a 96-well plate at a density of 104-105 cells/well in 120 µl of culture medium and grow the cells overnight in a CO2 incubator at 37°C. If you are using a culture medium containing serum, be sure to include a couple of wells containing culture medium only (i.e., without any cells). These will be your background controls. 2. The next day, treat the cells with or without compounds to be tested. We recommend that each treatment be performed at least in duplicate. 3. Culture the cells in a CO2 incubator at 37°C for 24 hours, or for a period of time according to your typical experimental protocol. PRE-ASSAY PREPARATION 7 Reagent Preparation Some of the kit components are in lyophilized or concentrated form and need to be reconstituted or diluted prior to use. Follow the directions carefully to ensure proper volumes of Assay Buffer are used to reconstitute or dilute the vial components. 1. Assay Buffer Preparation Dissolve the Cell-Based Assay Buffer Tablet (Item No. 10009322) in 100 ml of distilled water. This Buffer should be stable for approximately one year at room temperature. 2. Glycolysis Assay Enzyme Mixture - (Item No. 600452) The vial contains a lyophilized powder of enzymes. Immediately prior to use, reconstitute the contents of the vial with 150 µl of Assay Buffer. If you are not using all of the mixture at one time, make small aliquots and store at -80°C. Freezing and thawing of this solution should be limited to a single time. 3. Reaction Solution To make 12 ml of Reaction Solution sufficient for use on one 96-well plate, add 120 µl of each of the following mixtures to 11.64 ml of the Assay Buffer: Glycolysis Assay Substrate (Item No. 600451) ASSAY PROTOCOL Standard Preparation NOTE: Fetal bovine serum (FBS) contains LDH and thus will generate a high background reading when used in the culture medium. We recommend that you use culture medium containing a low percentage of FBS (such as 0.25-1%) or serum-free culture medium for your experiments. When culture medium containing FBS is used, wells containing the same culture medium without cells should be included in the assay as background controls. Glycolysis Assay L-Lactate Standard To prepare the standard for use in the glycolysis assay, obtain eight clean test tubes or microcentrifuge tubes and label them #1 through #8. Aliquot 450 µl of Assay Buffer into tube #1 and 250 µl into tubes #2-#8. Transfer 50 µl of the L-Lactic Acid Standard (Item No. 600453) into tube #1 and mix thoroughly. The lactic acid concentration of this standard, the first point on the standard curve, is 1 mM. Serially dilute the standard by removing 250 µl for tube #1 and place into tube #2, mix thoroughly. Next, remove 250 µl from tube #2 and place into tube #3; mix thoroughly. Repeat this procedure for tubes #4 through tubes #7. Do not add any standard to tube #8. This tube will be your blank. Glycolysis Assay Cofactor (Item No. 600454) 50 µl 250 µl 250 µl 250 µl 250 µl 250 µl 250 µl Reconstituted Glycolysis Assay Enzyme Mixture (prepared in Step 2) The Reaction Solution is stable for approximately one hour at room temperature. S1 S2 250 µl Assay Buffer 450 µl Assay Buffer 10 mM Bulk Standard 1 mM S3 500 µM S4 250 µl Assay Buffer 250 µM S5 250 µl Assay Buffer 125 µM S6 250 µl Assay Buffer 62.5 µM S7 250 µl Assay Buffer 31.3 µM S8 Final Blank 250 µl Assay Buffer 250 µl Assay Buffer 15.7 µM 0 µM Figure 1. Preparation of the L-Lactic Acid Standards 8 PRE-ASSAY PREPARATION ASSAY PROTOCOL 9 Plate Set Up Performing the Assay There is no specific pattern for using the wells on the plate. However, a lactate standard curve in duplicate has to be assayed with the samples. We suggest that each sample be assayed at least in duplicate. A typical layout of standards and samples is given below (see Figure 2). We suggest that you record the contents of each well on the template sheet provided (see page 19). 5 6 8 9 10 11 12 S9 S9 S17 S17 S17 S25 • Use different tips to pipette each reagent. • Before pipetting each reagent, equilibrate the pipette tip in that reagent (i.e., slowly fill the tip and gently expel the contents, repeat several times). • Do not expose the pipette tip to the reagent(s) already in the well. 1 2 3 4 A A A S1 S1 S1 B B B S2 S2 S2 S10 S10 S10 S18 S18 S18 S25 C C C S3 S3 S3 D D D S4 S4 S4 S12 S12 S12 S20 S20 S20 S26 3. Add 90 µl of Assay Buffer to each well except standard wells. E E E S5 S5 S5 S13 S13 S13 S21 S21 S21 S26 4. F F F S6 S6 S6 S14 S14 S14 S22 S22 S22 S26 S9 7 Pipetting Hints S11 S11 S11 S19 S19 S19 S25 G G G S7 S7 S7 H H H S8 S8 S8 S16 S16 S16 S24 S24 S24 S15 S15 S15 S23 S23 S23 A-H = Standards S1-S26 = Sample Wells 1. Centrifuge the 96-well cell culture plate at 1,000 rpm for five miutes. 2. Using a new 96-well plate, transfer 100 µl of the standards prepared above into appropriate wells. We recommend that the standards be run in duplicate. Transfer 10 µl of supernatant from each well of the cultured cell plate to corresponding wells on the new plate. (A smaller volume, 5 µl, of supernatant may be used if you anticipate a high rate of glycolysis and you are using the L-Lactic Acid Standard to calculate the level of glycolysis.) 5. Add 100 µl of Reaction Solution (prepared in Step 3, on page 8) to each well, including the standard wells, using a repeating pipettor. 6. Incubate the plate with gentle shaking on an orbital shaker for 30 minutes at room temperature. 7. Read the absorbance at 490 nm with a plate reader. Figure 2. Sample plate format 10 ASSAY PROTOCOL ASSAY PROTOCOL 11 The standard curve presented here is an example of the data typically produced with this assay. However, your results will not be identical to these. You must run a new standard curve with each experiment. ANALYSIS Calculations 0.5 1. Determine the average absorbance of each standard and sample. 2. Subtract the absorbance value of the blank from itself and all other standards and samples. This is the corrected absorbance. Plot the corrected absorbance values (from step 2 above) of each standard as a function of the final concentration of lactic acid. See Figure 3, on page 13, for a typical standard curve. 4. Calculate the L-lactate concentration of the samples using the the corrected absorbance of each sample and the equation below. L-Lactate (µM) = [ Absorbance - (y-intercept) Slope ] x 10* *Multiply by 20 if using 5 µl of culture supernatant. Performance Characteristics Specificity: To assess substrate specificity, the assay was performed with L-lactate replaced by structurally similar compound D-lactate. No reaction occurred when D-lactate is used at a concentration up to 10 mM. 12 ANALYSIS Slope = 0.4243 y-intercept = 0.0027 r2 = 0.9987 0.4 Absorbance (490 nM) 3. 0.3 0.2 0.1 0.0 0.0 0.2 0.4 0.6 0.8 1.0 1.2 L-Lactate (mM) Figure 3. L-Lactate Standard curve ANALYSIS 13 RESOURCES 9 8 Troubleshooting Lactic Acid Level (mM) 7 6 Problem Possible Causes Recommended Solutions 5 Erratic values; dispersion of duplicates A. Poor pipetting/technique B. Bubble in the well(s) A. Be careful not to splash the contents of the wells B. Carefully tap the side of the plate with your finger to remove bubbles No signal detected above background in the sample wells Sample was too dilute Re-assay the sample using a lower dilution The absorbance values of the sample wells were higher than the last standard Sample was too concentrated Re-assay the sample using a higher dilution 4 3 2 1 0 Control Insulin (1 µg/ml) Figure 4. Insulin stimulates glycolysis in HEK293 cells. HEK293 cells were seeded in a 96-well plate at a density of 50,000 cells/well in DMEM containing 10% FBS and grown in a 5% CO2 cell culture incubator at 37°C. The next day, cells were treated with vehicle or 1 µg/ml insulin in DMEM without FBS for 16 hours. Culture supernatant (10 µl) was removed from each well and the levels of lactic acid were assayed according to the protocol described in the booklet. Insulin at 1 µg/ml increased the production of extracellular lactic acid, indicating insulin stimulates glycolysis in HEK293 cells. 14 ANALYSIS RESOURCES 15 References Related Products 1. Demetrius, L.A., Coy, J.F., and Tuszynski, J.A. Cancer proliferation and therapy: The Warburg effect and quantum metabolism. Theoretical Biology and Medical Modeling 7, 2-14 (2010). 2. Griffin, J.L. and Shockcor, J.P. Metabolic profiles of cancer cells. Cancer 4, 551-561 (2004). 3. McCarty, M.F. and Whitaker, J. Manipulating tumor acidification as a cancer treatment strategy. Altern. Med. Rev. 15(3), 264-272 (2010). 4. Kondoh, H., Lleonart, M.E., Nakashima, Y., et al. A common metabolic profile shared between murine ES cells and primary cells bypassing senescence. Medical Hypothesis and Research 4(1), 29-36 (2008). 5. Barger, J.F. and Plas, D.R. Balancing biosynthesis and bioenergetics: Metabolic programs in oncogenesis. Endocrin-Related Cancer 17, R287-R304 (2010). Adipogenesis Assay Kit - Item No. 10006908 Adipolysis Assay Kit - Item No. 10009381 Alanine Transaminase Activity Assay Kit - Item No. 700260 Annexin V FITC Assay Kit - Item No. 600300 Chloride Colorimetric Assay Kit - Item No. 700610 Coenzyme A Assay Kit - Item No. 700440 Glucose Colorimetric Assay Kit - Item No. 10009582 Glutathione Cell-Based Detection Kit (Blue Fluorescence) - Item No. 600360 Glycogen Assay Kit - Item No. 700480 JC-1 Mitochondrial Membrane Potential Assay Kit - Item No. 10009172 D-Lactate Assay Kit - Item No. 700520 L-Lactate Assay Kit - Item No. 700510 LDH Cytotoxicity Assay Kit - Item No. 10008882 Lipase Activity Assay Kit - Item No. 700640 Multi-Drug Resistance Assay Kit (Calcein AM) - Item No. 600370 Multi-Parameter Apoptosis Assay Kit - Item No. 600330 NAD+/NADH Cell-Based Assay Kit - Item No. 600480 Oxygen Consumption/Glycolysis Dual Assay Kit - Item No. 601060 Oxygen Consumption/MitoMembrane Potential Dual Assay Kit - Item No. 600880 Oxygen Consumption Rate Assay Kit (MitoXpress®-Xtra HS Method) - Item No. 600800 20S Proteasome Assay Kit - Item No. 10008041 Pyruvate Assay Kit - Item No. 700470 WST-1 Cell Proliferation Assay Kit - Item No. 10008883 WST-8 Cell Proliferation Assay Kit - Item No. 10010199 XTT Cell Proliferation Assay Kit - Item No. 10010200 16 RESOURCES RESOURCES 17 18 RESOURCES RESOURCES H G F E A For further details, please refer to our Warranty and Limitation of Remedy located on our website and in our catalog. D Said refund or replacement is conditioned on Buyer giving written notice to Cayman within thirty (30) days after arrival of the material at its destination. Failure of Buyer to give said notice within thirty (30) days shall constitute a waiver by Buyer of all claims hereunder with respect to said material. C Buyer’s exclusive remedy and Cayman’s sole liability hereunder shall be limited to a refund of the purchase price, or at Cayman’s option, the replacement, at no cost to Buyer, of all material that does not meet our specifications. B Cayman Chemical Company makes no warranty or guarantee of any kind, whether written or oral, expressed or implied, including without limitation, any warranty of fitness for a particular purpose, suitability and merchantability, which extends beyond the description of the chemicals hereof. Cayman warrants only to the original customer that the material will meet our specifications at the time of delivery. Cayman will carry out its delivery obligations with due care and skill. Thus, in no event will Cayman have any obligation or liability, whether in tort (including negligence) or in contract, for any direct, indirect, incidental or consequential damages, even if Cayman is informed about their possible existence. This limitation of liability does not apply in the case of intentional acts or negligence of Cayman, its directors or its employees. 1 2 3 4 5 6 7 8 9 10 11 12 Warranty and Limitation of Remedy 19 NOTES This document is copyrighted. All rights are reserved. This document may not, in whole or part, be copied, photocopied, reproduced, translated, or reduced to any electronic medium or machine-readable form without prior consent, in writing, from Cayman Chemical Company. ©05/02/2014, Cayman Chemical Company, Ann Arbor, MI, All rights reserved. Printed in U.S.A. 20 RESOURCES