User Guide - Seahorse Bioscience
Contents
1. Final well uM 0 5 1 0 2 0 Final well uM 0 125 0 25 0 5 1 0 2 0 Final well uM 0 5 Constant Volume Starting well volume 525 uL assay medium Stock volume uL 120 240 480 Stock volume uL 33 8 67 5 135 270 540 Stock volume uL 300 Media volume uL 2 880 2 760 2 520 Media volume uL 2 966 2 2 932 5 2 865 2 730 2 460 Media volume uL 2 700 8X port uM 4 8 16 9X port uM 1 125 2 25 4 5 18 10X port uM 5 Add to port uL 75 75 75 Add to port uL 75 75 75 75 Add to port uL 75 Constant Concentration Starting well volume 500 uL assay medium Stock volume uL 150 300 600 Stock volume uL 37 5 75 150 300 600 Stock volume uL 300 Media volume uL 2 850 2 700 2 400 Media volume uL 2 962 5 2 925 2 850 2 700 2 400 Media volume uL 2 700 10X port uM 5 10 20 10X port uM 1 25 2 5 10 20 10X port uM 5 Refer to the Basic Procedure Loading the Sensor Cartridge with Compounds on the Seahorse Bioscience website for proper port loading technique www seahorsebio com resources documentation basicprocedures index php 1 Standard Assay no additional injection Load compounds into the appropriate ports of a hydrated sensor cartridge Port A Oligomycin Port B FCCP Port C Rotenone antimycin A 2 Modified Assay additional injecti2. Cell Mito Stress Test Reagents in order of injection Figure 1 XF Cell Mito Stress Test profile of the key parameters of mitochondrial respiration Sequential compound injections measure basal respiration ATP production proton leak maximal respiration spare respiratory capacity and non mitochondrial respiration Figure 2 XF Cell Mito Stress Test Modulators of the ETC This diagram illustrates the complexes of the ETC and the target of action of all of the compounds in the XF Cell Mito Stress Test Kit Oligomycin inhibits ATP synthase complex V FCCP uncouples oxygen consumption from ATP production and rotenone and antimycin A inhibit complexes and Ill respectively Compound s ETC Target Effect on OCR ATP Synthase Oligomycin Complex V Decrease FEER Inner Mitochondrial Membrane Increase Rotenone and Antimycin A Complex I and III respectively Decrease XF Cell Mito Stress Test User Guide 3 Seahorse Bioscience Kit Contents The XF Cell Mito Stress Test Kit includes 6 foil pouches that each contain reagents sufficient for a complete XF Cell Mito Stress Test in either the 96 or 24 well XF Cell Culture Microplate Every pouch includes one tube of each of the following compounds oligomycin FCCP and a mix of rotenone antimycin A as indicated below Compound Cap Color Quantity per tube nmol Blue 63 Yellow 72 Red 27 of both Kit Storage Product ships at ambient temperature Product should be stored at 20 C upo
3. Seahorse Bioscience website Visit www seahorsebio com support software stress test generator php to learn more about the XF Stress Test Report Generators and download the User Guide XF Cell Mito Stress Test User Guide 8 Seahorse Bioscience 103016 400 Rev A
4. User Guide XF Cell Mito Stress Test Kit Reorder number 103015 100 For Research Use Only Use with XF and XF Extracellular Flux Analyzers Seahorse Bioscience Product Description The XF Cell Mito Stress Test is the standard assay for measuring mitochondrial function in cells The XF Cell Mito Stress Test Kit is designed to evaluate mitochondrial function using the XF and XF Extracellular Flux Analyzers The kit contains the reagents required to determine key parameters of mitochondrial function Refer the Definitions Box for more details The ability to assess mitochondrial function has enabled researchers to advance their understanding of metabolism s key role in cellular physiology disease pathology and etiology This assay provides insight into the cause of mitochondrial dysfunction and an in depth understanding of metabolic pathways signals and phenotypes Introduction The XF Cell Mito Stress Test measures key parameters of mitochondrial function by directly measuring the oxygen consumption rate OCR of cells Figure 1 The XF Cell Mito Stress Test uses modulators of respiration that specifically target components of the electron transport chain ETC in the mitochondria to reveal key parameters of metabolic function The modulators oligomycin FCCP and a mix of rotenone and antimycin A are serially injected to measure ATP production maximal respiration and non mitochondrial respiration respectively Proton lea
5. and requires that each compound be prepared at a different concentration OR Table 3 describes how to prepare to load the cartridges using both options Prepare 3 mL of each compound in assay medium according to the approach you have chosen Seahorse recommends using 1 pM of oligomycin for most cells Please contact Technical Support with any questions Table 3 Compound Preparation for Loading Sensor Cartridge Ports XF XF 96 Constant Volume Constant Concentration Starting well volume 175 uL assay medium Starting well volume 180 uL assay medium Final well Stock Media 8X Addto Stock Media 10X Add to HM volume volume Port port volume volume Port port uL uL uM uL uL uL uM uL 0 5 120 2 880 4 25 150 2 850 5 20 1 0 240 2 760 8 25 300 2 700 10 20 2 0 480 2 520 16 25 600 2 400 20 20 Final well Stock Media 9X Addto Stock Media 10X Add to HM volume volume Port port volume volume Port port uL uL uM uL uL uL uM uL 0 125 33 8 2 966 2 1 125 25 37 5 2 962 5 1 25 22 0 25 67 5 2 932 5 2 25 25 75 2 925 25 22 0 5 135 2 865 4 5 25 150 2 850 5 22 1 0 270 2 730 9 25 300 2 700 10 22 2 0 540 2 460 18 25 600 2 400 20 22 Final well Stock Media 10X Addto Stock Media 10X Add to HM volume volume Port port volume volume Port port uL uL uM uL uL uL uM uL 0 5 300 2 700 5 25 300 2 700 5 25 XF Cell Mito Stress Test User Guide 6 Seahorse Bioscience XF XF 24 Load Sensor Cartridge
6. ions to cover the desired concentration range Important Use compounds the same day they are reconstituted Do not refreeze Discard any remaining compound 1 Remove foil pouch from XF Cell Mito Stress Test Kit box Each pouch contains reagents sufficient for a complete XF Cell Mito Stress Test in a 96 or 24 well XF Cell Culture Microplate 2 Allow compounds to warm to room temp in the sealed pouch for approximately 15 minutes 3 Open pouch and remove the three tubes containing oligomycin blue cap FCCP yellow cap and rotenone antimycin A red cap Place tubes in a small tube rack 4 Resuspend contents of each tube with prepared assay medium in volumes described in Table 2 with a p1000 pipette Gently pipette up and down 10 times to solubilize the compounds XF Cell Mito Stress Test User Guide 5 Seahorse Bioscience Table 2 Stock Solutions Volume of Assay Medium Final Concentration 630 uL 100 yM 720 uL 100 yM 540 uL 50 uM Prepare Compounds for Loading in Sensor Cartridge There are two approaches to loading the injection ports of the sensor cartridge Constant compound concentration variable loading volume This approach entails preparing the compounds at a constant concentration and requires that a different volume of each compound be loaded in the injection port Constant loading volume variable compound concentration This approach entails loading a constant volume of compound in each injection port
7. iresatciinele 3 Hydrate a sensor cartridge in XF Calibrant at 37 C in a non COz2 incubator on the Seahorse website for more overnight Refer to Basic Procedure Hydrating the Sensor Cartridge on f i A aE information on optimization the Seahorse website for more details www seahorsebio com resources documentation basicprocedures The Cell Line Reference Database 4 Design experiment in Wave or XF software Use the default software i a www seahorsebio com learning cell settings See the instrument user manual for additional details line php is a good resource for finding Day of Assay reference publications for your cell type Prepare Assay Medium 1 4 Prepare assay medium by supplementing XF Base Medium Seahorse recommends 1 mM pyruvate 2 mM glutamine and 10 mM glucose as a starting point however desired medium composition can be varied depending on cell type or in vitro culture conditions Refer to Basic Procedure Preparing Assay Media for Use XF Assays on the Seahorse Bioscience website for more information www seahorsebio com resources documentation basicprocedures Warm assay medium to 37 C Adjust pH to 7 4 with NaOH Note Seahorse recommends sterile filtration following pH adjustment Keep at 37 C until ready to use Prepare Stock Compounds of interest If you are optimizing concentrations for your assay start with the highest concentrations given in Table 3 and make serial dilut
8. k and spare respiratory capacity are then calculated using these parameters and basal respiration As illustrated in Figure 2 and described in Table 1 each modulator targets a specific component of the ETC Oligomycin inhibits ATP synthase complex V and the decrease in OCR following injection of oligomycin correlates to the mitochondrial respiration associated with cellular ATP production Carbonyl cyanide 4 trifluoromethoxy phenylhydrazone FCCP is an uncoupling agent that collapses the proton gradient and disrupts the mitochondrial membrane potential As a result electron flow through the ETC is uninhibited and oxygen is maximally consumed by complex IV The FCCP stimulated OCR can then be used to calculate spare respiratory capacity defined as the difference between maximal respiration and basal respiration Spare respiratory capacity is a measure of the cell s ability to respond to increased energy demand The third injection is a mix of rotenone a complex inhibitor and antimycin A a complex III inhibitor This combination shuts down mitochondrial respiration and enables the calculation of non mitochondrial respiration driven by processes outside the mitochondria XF Cell Mito Stress Test User Guide 2 Seahorse Bioscience OCR pMoles min Mitochondrial Respiration Oligomycin FCCP AntimycinA amp Rotenone 0 10 20 30 40 50 60 70 80 90 100 110 TIME minutes Intermembrane space Mitochondrial matrix Table 1 XF
9. n arrival Additional Required Items XF XF96 or24 Analyzer Seahorse Bioscience XF Base Medium Seahorse Bioscience 102353 100 100 mM Pyruvate Sigma S8636 or equivalent 200 mM Glutamine Sigma G8540 or equivalent 2 5 M Glucose Sigma G8769 or equivalent Narrow p1000 pipette tips are recommended for reconstituting compounds within the tubes provided e g Fisherbrand SureOne Micropoint Pipet Tips catalog 02 707 402 Figure 3 XF Cell Mito Stress Test Assay Workflow Prior to Assay XF Cell Mito Stress Test User Guide 4 GLOSSARY Basal Respiration Oxygen consumption used to meet cellular ATP demand and resulting from mitochondrial proton leak Shows energetic demand of the cell under baseline conditions ATP Production The decrease in oxygen consumption rate upon injection of the ATP synthase inhibitor oligomycin represents the portion of basal respiration that was being used to drive ATP production Shows ATP produced by the mitochondria that contributes to meeting the energetic needs of the cell H Proton Leak Remaining basal respiration not coupled to ATP production Proton leak can be a sign of mitochondrial damage or can be used as a mechanism to regulate the mitochondrial ATP production Maximal Respiration The maximal oxygen consumption rate attained by adding the uncoupler FCCP FCCP mimics a physiological energy demand by stimulating the respiratory chain to operate at maximum ca
10. on included Add to port uL 56 56 56 Add to port uL 62 62 62 62 62 Add to port uL 69 To inject an additional compound prior to oligomycin use port A for the desired compound and then load Port B Oligomycin Port C FCCP Port D Rotenone antimycin A Table 4 lists the appropriate volumes and concentrations for this injection scheme XF Cell Mito Stress Test User Guide Seahorse Bioscience Table 4 Compound Injection Volumes Involving an Acute Injection XFe XF 96 XFe XF 24 Constant Volume Constant Constant Volume Constant Concentration Starting well volume Concentration Starting well volume Starting well volume 175 uL assay medium Starting well volume 525 uL assay medium 500 uL assay medium 180 uL assay medium Port Vol Conc Vol Conc Vol Conc Vol Conc Vol A 25 uL 8X 20 uL 10X 75 uL 8X 56 uL 10X B 25 uL 9X 22 uL 10X 75 uL 9X 62 uL 10X c 25 uL 10X 25 uL 10X 75 uL 10X 69 uL 10X D 25 uL 11X 27 uL 10X 75 uL 11X 75 uL 10X Prepare XF Cell Culture Microplate for Assay 1 Remove cell culture microplates from 37 C CO2 incubator and examine cells under microscope to confirm confluence Remove assay medium from water bath 3 Change the cell culture growth medium in the cell culture microplate to warmed assay medium using a multichannel pipette and place the cell culture microplate into a 37 C non COz2 incubator for 45 minutes to 1 hour prior to the assay Run the XF Cell Mito Stress Test O
11. pacity which causes rapid oxidation of substrates sugars fats amino acids to meet this metabolic challenge Shows the maximum rate of respiration that the cell can achieve Spare Respiratory Capacity This measurement indicates the capability of the cell to respond to an energetic demand as well as how closely the cell is to respiring to its theoretical maximum The cell s ability to respond to demand can be an indicator of cell fitness or flexibility Non mitochondrial Respiration Oxygen consumption that persists due to a subset of cellular enzymes that continue to consume oxygen after rotenone and antimycin A addition This is important for getting an accurate measure of mitochondrial respiration Seahorse Bioscience Workflow Day Prior to Assay A NOTE ON OPTIMIZATION Optimal concentrations of FCCP and cell 1 Turn on the XF XF Analyzer and warm up overnight minimum 5 hours seeding density should be empirically 2 Plate cells at a previously determined optimized density in the XF Cell determined for your cells prior to the Culture Microplate using the appropriate cell culture growth medium assay Refer to FCCP Optimization with Refer to Basic Procedure Seeding Cells in XF Cell Culture Microplates the XF Cell Mito Stress Test and available on the Seahorse Bioscience website for more information Seeding Cells in XF Cell Culture www seahorsebio com resources documentation basicprocedures Rilerapiates Bele cced
12. pen software and retrieve saved assay template file Follow instructions below for your specific software If you are using XF Software 1 Browse for and open the saved design file then click Run 2 Place the calibration plate with the loaded sensor cartridge on the instrument tray Calibration will take approximately 15 30 minutes Note Remove cartridge lid and verify correct plate orientation 3 When prompted replace the calibration plate with the cell culture microplate then click Start If you are using Wave 1 Browse and open the saved design file select the Review and Run tab and then click Start Run 2 When prompted place the loaded sensor cartridge with the calibrant plate into the instrument then click I m ready Calibration will take approximately 15 30 minutes Note Remove cartridge lid and verify correct plate orientation 3 Following calibration and equilibration of the cell culture microplate when prompted click I m ready Load the cell culture microplate and click I m ready to run the assay Data Analysis The XF Mito Stress Test Report Generator automatically calculates the XF Cell Mito Stress Test parameters from Wave data that has been exported to Excel It can be used with either a standard or modified stress test protocol and provides a convenient customizable one page assay summary The Report Generator can be installed either alongside Wave or directly from the
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