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Agilent Seahorse XFp Glycolytic Rate Assay Kit User Guide

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Contents

Introduction

Assay Background

9

Kit Information

Kit Shipping and Storage

11

Additional Required Items

12

Assay Workflow

Day Prior to Assay

14

Running the Assay

18

Analysis Using the Agilent Seahorse Glycolytic Rate Assay Report

19

Frequently Asked Questions

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Содержание Seahorse XFp

Страница 1: ...Agilent Technologies Agilent Seahorse XFp Glycolytic Rate Assay Kit User Guide Kit 103346 100...

Страница 2: ...ibed in this document are furnished under a license and may be used or copied only in accordance with the terms of such license Restricted Rights Legend If software is for use in the performance of a...

Страница 3: ...ckground 5 Glossary 9 Kit Information Kit Contents 11 Kit Shipping and Storage 11 Additional Required Items 12 Assay Workflow Day Prior to Assay 14 Day of Assay 14 Running the Assay 18 Analysis Using...

Страница 4: ...4 Agilent Seahorse XFp Glycolytic Rate Assay Kit User Guide...

Страница 5: ...reliable method for measuring glycolysis in cells Together with a Seahorse XFp Analyzer the Seahorse XFp Glycolytic Rate Assay provides accurate measurements of glycolytic rates for basal conditions...

Страница 6: ...PER of the cells defined below Glucose in cells is converted to pyruvate and then converted to lactate in the cytoplasm or to CO2 and water in the mitochondria The conversion of glucose to lactate res...

Страница 7: ...xtruded into the extracellular medium during glycolysis This assay allows for real time measurements of changes in glycolysis rates that may go undetected in long term lactate accumulation assays The...

Страница 8: ...ular media which is detected by the XFp Analyzer as ECAR Additionally mitochondrial TCA activity produces CO2 which hydrates and acidifies the media By inhibiting respiration OCR during the assay with...

Страница 9: ...extracellular lactate production rate Compensatory glycolysis The rate of glycolysis in cells following the addition of mitochondrial inhibitors effectively inhibiting oxidative phosphorylation and d...

Страница 10: ...10 Agilent Seahorse XFp Glycolytic Rate Assay Kit User Guide Introduction...

Страница 11: ...mperature The product is stable for one year from the date of manufacture The actual expiration date is printed on the label of the assay kit box Depending on the shipping date the actual shelf life o...

Страница 12: ...cartidges miniplates and calibrant Agilent Technologies 103022 100 XF DMEM medium pH 7 4 or XF RPMI medium pH 7 4 XF DMEM or RPMI media can also be purchased together with the supplements listed in th...

Страница 13: ...t User Guide Agilent Technologies 3 Assay Workflow Day Prior to Assay 14 Day of Assay 14 Running the Assay 18 Analysis Using the Agilent Seahorse Glycolytic Rate Assay Report Generator 19 Figure 3 Agi...

Страница 14: ...No 103575 100 and 103576 100 These media contain the appropriate amount of HEPES and NO additional HEPES is needed It is recommended to start with 1 mM pyruvate 2 mM glutamine and 10 mM glucose Howeve...

Страница 15: ...say for details Remove the cell culture growth medium in the cell culture miniplate Wash once with warmed assay medium using a multichannel pipette and incubate with assay medium at 37 C in a non CO2...

Страница 16: ...s on the bottom of the tube Vortex 1 minute to ensure that compounds go into solution Dilute compounds Table 3 describes how to prepare compounds dilutions to load the cartridge Please note that if di...

Страница 17: ...rior to the Glycolytic Rate Assay compounds use port A for the desired compound and then load ports as follows Port A experimental compound acute injection or media control Port B Rot AA Port C 2 DG T...

Страница 18: ...rted then click Import 7 All imported Assay Templates will now be available for selection from the Local tab when starting an XFp assay Run the Seahorse XF Glycolytic Rate Assay 1 Click Start 2 Select...

Страница 19: ...save your assay file asyr 2 In your Desktop version of Wave 2 3 or higher open the file from your USB Flash Drive 3 From Wave export the data from the completed run as an xls file 4 Load the data fil...

Страница 20: ...20 Agilent Seahorse XFp Glycolytic Rate Assay Kit User Guide Assay Workflow...

Страница 21: ...nd using the Buffer Factor BF and CO2 contribution factor CCF the mitochondrial PER is calculated and subtracted to generate the glycoPER The Seahorse XF Glycolytic Rate Assay reports the percentage o...

Страница 22: ...chondrial sources of acidification as well as reporting the data in standard units pmol min These features make the Seahorse XF Glycolytic Rate Assay highly comparable to extracellular lactate product...

Страница 23: ......

Страница 24: ...Agilent Technologies For research use only Not for use in diagnostic procedures Agilent Technologies Inc Printed in USA May 2019 Revision B0 103346 400 103346 400...

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