ab Glucose Uptake Assay Kit (Colorimetric)

Transcription

1 Version 15c Last updated 8 August 2017 ab Glucose Uptake Assay Kit (Colorimetric) For the rapid, sensitive and accurate measurement of glucose uptake in cells. This product is for research use only and is not intended for diagnostic use. Copyright 2017 Abcam. All rights reserved

2 Table of Contents 1. Overview 1 2. Protocol Summary 2 3. Precautions 3 4. Storage and Stability 3 5. Limitations 4 6. Materials Supplied 4 7. Materials Required, Not Supplied 5 8. Technical Hints 6 9. Reagent Preparation Standard Preparation Assay Procedure Calculations Typical Data Troubleshooting FAQs Notes 19 Copyright 2017 Abcam. All rights reserved

3 1. Overview Glucose Uptake Assay Kit (Colorimetric) (ab136955) is an assay that uses the glucose analog 2-deoxyglucose (2-DG) to detect and quantify glycose uptake in cells. This easy to use, non-radioactive kit is highly sensitive and can detect as low as 10 pmol 2-DG uptake per well. Among many different methods available for measuring glucose uptake, 2-deoxyglucose (2-DG) has been widely used because of its structural similarity to glucose. Like glucose, 2-DG can be taken up by glucose transporters and metabolized to 2-DG-6- phosphate (2-DG6P). However, 2-DG6P cannot be metabolized further and thus accumulates in the cells. The accumulated 2-DG6P is oxidized to generate NADPH, which results in oxidation of a substrate. The oxidized substrate can then be detected at OD=412 nm. The amount of accumulated NAPDH, and therefore 2-DG6P, is directly proportional to 2-DG (or glucose) taken up by cells. Figure 1. Assay procedure. 2-DG oxidation generates NADPH (step A), which leads to production of an oxidized product that can be detected at OD 412 nm (step B). Glucose uptake is an important biological process for studying cell signaling and glucose metabolism. This product can be used for the measurement of glucose uptake in response to insulin, growth factors, cytokines, mitogens or nutrients, as well as to analyze glucose metabolism and signaling in various cell types and the screening of anti-diabetic drugs. ab Glucose Uptake Assay Kit (colorimetric) 1

4 2. Protocol Summary Grow cells in serum free medium and incubate O/N Wash cells and incubate in KRPH/2% BSA for 40 minutes Add stimulator +/- inhibitor together with 2-DG to cells Prepare 2-DG6P standard curve Degradation of endogenous NAD(P) Generation of NADPH from 2DG6P Degradation of unused NAD(P) Recycling amplification reaction Measure uptake at OD412 nm in a kinetic mode ab Glucose Uptake Assay Kit (colorimetric) 2

5 3. Precautions Please read these instructions carefully prior to beginning the assay. All kit components have been formulated and quality control tested to function successfully as a kit. We understand that, occasionally, experimental protocols might need to be modified to meet unique experimental circumstances. However, we cannot guarantee the performance of the product outside the conditions detailed in this protocol booklet. Reagents should be treated as possible mutagens and should be handled with care and disposed of properly. Please review the Safety Datasheet (SDS) provided with the product for information on the specific components. Observe good laboratory practices. Gloves, lab coat, and protective eyewear should always be worn. Never pipet by mouth. Do not eat, drink or smoke in the laboratory areas. All biological materials should be treated as potentially hazardous and handled as such. They should be disposed of in accordance with established safety procedures. 4. Storage and Stability Store kit at -20 C in the dark immediately upon receipt. Kit has a storage time of 1 year from receipt, providing components have not been reconstituted. Refer to list of materials supplied for storage conditions of individual components. Observe the storage conditions for individual prepared components in the Materials Supplied section. Aliquot components in working volumes before storing at the recommended temperature. Note: Reconstituted components are stable for 2 months. ab Glucose Uptake Assay Kit (colorimetric) 3

6 5. Limitations Assay kit intended for research use only. Not for use in diagnostic procedures. Do not mix or substitute reagents or materials from other kit lots or vendors. Kits are QC tested as a set of components and performance cannot be guaranteed if utilized separately or substituted. 6. Materials Supplied Item Quantity Storage Condition (before prep) Storage Condition (after prep) Assay Buffer 25 ml -20 C -20 C Extraction Buffer 17 ml -20 C -20 C Neutralization Buffer 2.5 ml -20 C -20 C Enzyme mix (lyophilized) 1 vial -20 C -20 C Glutathione Reductase (lyophilized) 2 vials -20 C -20 C Recycling Mix (lyophilized) 1 vial -20 C -20 C Substrate DTNB (lyophilized) 2 vials -20 C -20 C 2-Deoxyglucose (2-DG), 10 mm 1 ml -20 C -20 C 2-DG6P standard (lyophilized) 1 vial -20 C -20 C ab Glucose Uptake Assay Kit (colorimetric) 4

7 7. Materials Required, Not Supplied These materials are not included in the kit, but will be required to successfully perform this assay: Microplate reader capable of measuring absorbance at OD 412 nm MilliQ water or other type of double distilled water (ddh 2 O) Pipettes and pipette tips, including multi-channel pipette Assorted glassware for the preparation of reagents and buffer solutions Tubes for the preparation of reagents and buffer solutions Sterile 96 well plate with clear flat bottom Plate sealing tape General tissue culture supplies PBS Bovine Serum Albumin (BSA) KRPH (Krebs-Ringer-Phosphate-Hepes) buffer: 20 mm HEPES, 5 mm KH 2 PO 4, 1 mm MgSO 4, 1 mm CaCl 2, 136 mm NaCl, 4.7 mm KCl, ph 7.4 Adipocyte culture medium (serum free and serum supplemented) if using a different cell type, use appropriate culture medium Insulin (glucose transporter activator) ab Glucose Uptake Assay Kit (colorimetric) 5

8 8. Technical Hints This kit is sold based on number of tests. A test simply refers to a single assay well. The number of wells that contain sample, control or standard will vary by product. Review the protocol completely to confirm this kit meets your requirements. Please contact our Technical Support staff with any questions. Selected components in this kit are supplied in surplus amount to account for additional dilutions, evaporation, or instrumentation settings where higher volumes are required. They should be disposed of in accordance with established safety procedures. Avoid foaming or bubbles when mixing or reconstituting components. Avoid cross contamination of samples or reagents by changing tips between sample, standard and reagent additions. Ensure plates are properly sealed or covered during incubation steps. Ensure all reagents and solutions are at the appropriate temperature before starting the assay. Samples generating values that are greater than the most concentrated standard should be further diluted in the appropriate sample dilution buffer. Make sure all necessary equipment is switched on and set at the appropriate temperature. ab Glucose Uptake Assay Kit (colorimetric) 6

9 9. Reagent Preparation Briefly centrifuge small vials at low speed prior to opening. 9.1 Assay Buffer: Ready to use as supplied. Equilibrate to room temperature before use. Store at -20 C. 9.2 Extraction Buffer: Ready to use as supplied. Equilibrate to room temperature before use. Store at -20 C. 9.3 Neutralization Buffer: Ready to use as supplied. Equilibrate to room temperature before use. Store at -20 C. 9.4 Enzyme Mix: Reconstitute with 220 µl Assay Buffer. Pipette up and down to dissolve completely. Aliquot enzyme mix so that you have enough volume to perform the desired number of assays. Store at -20 C. Avoid repeated freeze/thaw cycles. Once reconstituted, use within 2 months. 9.5 Glutathione Reductase: Reconstitute with 1.1 ml Assay Buffer. Pipette up and down to dissolve completely. Aliquot enzyme so that you have enough volume to perform the desired number of assays. Store at - 20 C. Avoid repeated freeze/thaw cycles. Once reconstituted, use within 2 months. 9.6 Recycling Mix: Reconstitute with 220 µl Assay Buffer. Pipette up and down to dissolve completely. Aliquot enzyme mix so that you have enough volume to perform the desired number of assays. Store at -20 C. Avoid repeated freeze/thaw cycles. Once reconstituted, use within 2 months. 9.7 Substrate DNTB: Reconstitute with 1 ml Assay Buffer. Pipette up and down to dissolve completely. Aliquot substrate so that you have enough volume to perform the desired number of assays. Store at -20 C. Avoid repeated freeze/thaw cycles. Once reconstituted, use within 2 months. ab Glucose Uptake Assay Kit (colorimetric) 7

10 9.8 2-Deoxyglucose (2-DG) (10 mm): Ready to use as supplied. Equilibrate to room temperature before use. Store at -20 C DG6P Standard: Reconstitute with 100 µl ddh 2 O to generate a 10 mm (10 nmol/µl) 2-DG6P solution. Pipette up and down to dissolve completely. Aliquot standard so that you have enough volume to perform the desired number of assays. Store at - 20 C. Avoid repeated freeze/thaw cycles. Once reconstituted, use within 2 months. Keep on ice while in use. ab Glucose Uptake Assay Kit (colorimetric) 8

11 10. Standard Preparation Always prepare a fresh set of standards for every use. Discard working standard dilutions after use as they do not store well. Standard dilution can be prepared during insulin incubation step (Step ) 10.1 Prepare 500 µl of 0.1 mm (100 pmol/µl) 2-DG6P Standard by diluting 5 µl of the 10 mm 2-DG6P Standard in 495 µl Assay Buffer and mix well Prepare 500 µl of 0.01 mm (10 pmol/µl) by diluting 50 µl of 0.1 mm 2-DG6P standard (section 10.1) in 450 µl Assay Buffer Using 0.01 mm 2-DG6P standard, prepare standard curve dilution as described in the table in a microplate or microcentrifuge tubes: Standard # 2-DG6P Standard (µl) Assay Buffer (µl) Final volume standard in well (µl) End amount 2-DG6P in well (pmol/well) Each dilution has enough amount of standard to set up duplicate readings (2 x 50 µl). ab Glucose Uptake Assay Kit (colorimetric) 9

12 11. Assay Procedure Equilibrate all materials and prepared reagents to room temperature prior to use. We recommend that you assay all standards, controls and samples in duplicate. Prepare all reagents, working standards, and samples as directed in the previous sections. This protocol has been optimized for 3T3-L1 adipocytes. For other cell types, optimal incubation times and treatment protocols may vary from these conditions and should be set by the researcher. As initial recommendation, we suggest seeding cells at a density of 60 80% Seed cells: Seed cells at a density of ~ 1,500 3T3-L1 cells/well in 100 µl culture medium in a sterile 96-well plate and differentiate to mature adipocytes. Maintain differentiated cells for another 4 days prior to use. Note: Prepare enough cells to be able to set up control (untreated) and treated cells Wash adipocytes twice with PBS and starve in 100 µl serum free adipocyte medium overnight to increase glucose uptake Next day, wash cells 3X in PBS Starve cells for glucose by pre-incubating them with 100 µl KRPH/2% BSA buffer for 40 minutes Prepare samples: Control (untreated) cells: Wash cells 3X with PBS. Proceed to Step Treated cells: stimulate cells in KRPH + 2% BSA buffer with 1 µm insulin for 20 minutes to activate glucose transporter. Total volume well = 100 µl. Note: This incubation provides a quick and stimulation of glucose uptake via GLUT transporters Add 10 µl of 10 mm 2-DG (Step 9.8) to insulin treated cells and no insulin control; mix by pipetting up and down Incubate cells for 20 minutes. Note: 2-DG6P Standard dilution (Step 10.3) can be prepared during the insulin incubation. ab Glucose Uptake Assay Kit (colorimetric) 10

13 Wash cells 3X with PBS to remove exogenous 2-DG Endogenous NAD(P) degradation: Note: This step will degrade endogenous NAD(P) and denature enzymes in the samples to minimize background signal Lyse cells with 80 µl Extraction Buffer by pipetting up and down to release cells. If using a bigger culture surface, you can do this step with a cell scraper. Note: At this point, samples can be transferred to microcentrifuge tubes for ease of handling. They can also be left on the microplate if preferred Freeze/thaw cell lysates once and heat at 85 C for 40 minutes. Note: If you have used a plate and it has been covered with film or lid, you might find some condensation has formed. Spin the plate briefly (~ 500 rpm 1 minute) to get rid of the condensation bubbles before you take the lid or the cover off Cool cell lysate on ice for 5 minutes Neutralize sample by adding 10 µl of Neutralization Buffer. Briefly spin the samples to ensure proper mixing of reagents (~ 500 rpm for 1 2 minutes, or using soft spin cycle if available) Transfer supernatant to new tubes. Note: Pausing in the middle of the assay is not recommended. However, if really necessary, we suggest you freeze your samples at -80 after this step as all proteins will have been removed. Thaw samples on ice when you are ready to proceed with the assay Reaction wells set up: Samples (control sample and treated): dilute samples 1:10 to a total volume of 50 µl (5 µl sample + 45 Assay Buffer). Note: We recommend performing several different sample dilutions with Assay Buffer to ensure the readings fall within the standard curve. Standard wells = 50 µl standard dilutions. Control sample wells (no insulin treatment) = 1-50 µl diluted 1:10 sample/well (adjust to 50 µl/well with Assay Buffer). Treated sample wells: 1-50 µl diluted 1:10 sample/well (adjust to 50 µl/well with Assay Buffer). ab Glucose Uptake Assay Kit (colorimetric) 11

14 11.5 Assay procedure: Prepare 10 µl of Reaction Mix A (NADPH generation) for each reaction. Mix enough reagents for the number of assays (samples and controls) to be performed. Prepare a master mix of the Reaction mix to ensure consistency. Component Reaction Mix A (µl) Assay Buffer 8 Enzyme Mix Add 10 µl of Reaction Mix into each well of standard, control and sample wells Mix and incubate at 37 C for 1 hour Add 90 µl Extraction Buffer to each well, seal the microplate with a sealing tape and heat it at 85 C for 40 minutes to degrade any unused NADP left in the sample Cool plate on ice for 5 minutes and neutralize reaction by adding 12 µl of Neutralization Buffer Prepare 38 µl of Reaction Mix B (recycling amplification reaction) for each reaction. Mix enough reagents for the number of assays (samples and controls) to be performed. Prepare a master mix of the Reaction Mix to ensure consistency. Component Reaction Mix B (µl) Glutathione Reductase 20 Substrate DRNB 16 Recycling Mix Add 38 µl Reaction Mix B to each well (standard, control and treated cells) and mix well by pipetting up and down Plate measurement: Measure output at OD 412 nm on a microplate reader in a kinetic mode, every 2 3 minutes, at 37 C protected from light, until Standard #6 (100 pmol/well) reaches OD = Take an additional endpoint reading of all samples and standards. ab Glucose Uptake Assay Kit (colorimetric) 12

15 12. Calculations Samples producing signals greater than that of the highest standard should be further diluted in appropriate buffer and reanalyzed, then multiply the concentration found by the appropriate dilution factor Subtract the sample background control (cells not treated with insulin nor 2-DG) from sample reading Subtract the mean absorbance value of the blank (Standard #1) from all standard and sample readings. This is the corrected absorbance Average the duplicate reading for each standard and sample Plot the corrected absorbance values for each standard as a function of the final concentration of 2-DG6P Draw the best smooth curve through these points to construct the standard curve. Most plate reader software or Excel can plot these values and curve fit. Calculate the trendline equation based on your standard curve data (use the equation that provides the most accurate fit) Concentration of 2-DG in the test samples is calculated as: Where: 2 - DG uptake = ( Ts Sv) * D = pmol µl = nmol ml = µm Ts = amount of 2-DG6Pin the sample well calculated from standard curve (pmol). Sv = sample volume added in the sample wells (µl). D = sample dilution factor. ab Glucose Uptake Assay Kit (colorimetric) 13

16 13. Typical Data Typical standard curve data provided for demonstration purposes only. A new standard curve must be generated for each assay performed. Figure 2. Typical standard calibration curve (a) and 2-DG uptake in a variety of cell lines: 3T3-L1 cells (b), human adipocytes (c) and HeLa cells (d). I: insulin; P: phloretin (glucose transport inhibitor). ab Glucose Uptake Assay Kit (colorimetric) 14

17 Figure 3. Glucose uptake of 2-DG in 3T3-L1 adipocytes stimulated with insulin (I) compared with untreated cells. ab Glucose Uptake Assay Kit (colorimetric) 15

18 14. Troubleshooting Problem Reason Solution Assay not working Sample with erratic readings Lower/higher readings in samples and standards Use of ice-cold buffer Plate read at incorrect wavelength Use of a different microplate Cells/tissue samples not homogenized completely Samples used after multiple free/ thaw cycles Use of old or inappropriately stored samples Presence of interfering substance in the sample Improperly thawed components Allowing reagents to sit for extended times on ice Incorrect incubation times or temperatures Buffers must be at assay temperature Check the wavelength and filter settings of instrument Colorimetric: clear plates Fluorometric: black wells/clear bottom plates Luminometric: white wells/clear bottom plates Use Dounce homogenizer, increase number of strokes Aliquot and freeze samples if needed to use multiple times Use fresh samples or store at - 80 C (after snap freeze in liquid nitrogen) till use Check protocol for interfering substances; deproteinize samples Thaw all components completely and mix gently before use Always thaw and prepare fresh reaction mix before use Verify correct incubation times and temperatures in protocol ab Glucose Uptake Assay Kit (colorimetric) 16

19 Problem Reason Solution Standard readings do not follow a linear pattern Unanticipated results Pipetting errors in standard or reaction mix Air bubbles formed in well Standard stock is at incorrect concentration Measured at incorrect wavelength Samples contain interfering substances Sample readings above/ below the linear range Avoid pipetting small volumes (< 5 µl) and prepare a master mix whenever possible Pipette gently against the wall of the tubes Always refer to dilutions described in the protocol Check equipment and filter setting Troubleshoot if it interferes with the kit Concentrate/ Dilute sample so it is within the linear range ab Glucose Uptake Assay Kit (colorimetric) 17

20 15. FAQs Q. Can I start working on a 3.5 cm petri dish and then continue the assay in a 96-well plate after the lysis step? A. Yes, you can. You have to increase the volumes for all reagents used to ensure all cells are covered and not exposed to the air to dry out. Use the same amount of reagents that you would to culture cells for example, for the serum starvation incubation, instead of using 100 µl media for covering cells in the 96 well plate, you could use about 2 ml media for a 3.5 cm plate. Q. What is the purpose of starvation with serum free medium if it is then followed by starvation with glucose free buffer? A. Serum starvation is required because serum can provide a potential carbon source from which cells can then make glucose. Q. I need to culture my cells with 1% insulin. Should the insulin be removed during either serum starvation step and/or the glucose starvation stage? A. There should be no insulin in the media during starvation. After starvation, cells are stimulated with insulin to induce uptake of glucose via the GLUT transporters. ab Glucose Uptake Assay Kit (colorimetric) 18

21 16. Notes ab Glucose Uptake Assay Kit (colorimetric) 19

22 ab Glucose Uptake Assay Kit (colorimetric) 20

23 ab Glucose Uptake Assay Kit (colorimetric) 21

24 Technical Support Copyright 2016 Abcam, All Rights Reserved. The Abcam logo is a registered trademark. All information / detail is correct at time of going to print. Austria wissenschaftlicherdienst@abcam.com France supportscientifique@abcam.com Germany wissenschaftlicherdienst@abcam.com Spain soportecientifico@abcam.com Switzerland technical@abcam.com Deutsch: Français: UK, EU and ROW technical@abcam.com +44(0) Canada ca.technical@abcam.com US and Latin America us.technical@abcam.com Asia Pacific hk.technical@abcam.com (852) China cn.technical@abcam.com Japan technical@abcam.co.jp +81-(0) Singapore sg.technical@abcam.com Australia au.technical@abcam.com +61-(0) New Zealand nz.technical@abc.com +64-(0) Copyright 2017 Abcam. All rights reserved