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XF24 Training Manual

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Summary of Contents for XF24

Page 1: ...XF24 Training Manual 1...

Page 2: ...uxPak 18 100867 100 XF24 FluxPak mini 6 101174 100 XF24 Islet Capture FluxPak 6 XF Microplates Part Description of Microplates 100777 004 XF24 V7 PS Cell Culture Microplates 10 101122 100 XF24 Islet C...

Page 3: ...s must be loaded with either compound or assay medium EXAMPLE VOLUMES FOR AN XF ASSAY Number of compound injec tions in assay 1 2 3 4 XF Assay Medium in wells after medium exchange 525 900 L 525 825 L...

Page 4: ...ideos available at http www seahorsebio com learning videos php a Energy Pathways 2 06 minutes b Transient Microchamber 2 12 minutes c Drug Injection Ports 1 51 minutes d Validation 1 2 23 minutes e V...

Page 5: ...iments Planning Your Assay Obtain materials Reconstitute kits Review media requirements Day Prior to Assay Hydrate cartridge Seed cells Day of Assay Prepare and run assay Prepare assay medium Wash cel...

Page 6: ...pport Sample XF Assay Workflow Diagram Seed cells and incubate overnight in growth medium Hydrate sensor cartridge overnight Day Before XF Assay Day of XF Assay Change medium to bicarbonate free low b...

Page 7: ...P1000 Pipette tips Low buffered non bicarbonated Medium for Assay e g DMEM KHB RPMI without bicarbonate Recommended The following are materials recommended to help you achieve optimal results These i...

Page 8: ...for your experiments please visit our website http www seahorsebio com or contact Technical Support http www seahorsebio com support tech support php XF Prep Station Part 100900 100 XF Medium requires...

Page 9: ...cell suspension per well as shown in figure below do not seed cells in background correction wells A1 B4 C3 D6 Be sure to put medium only no cells in the background correction wells Allow plate to re...

Page 10: ...cific and may need to be determined empirically For the XF Cell Mito Stress Test assay medium with user added glucose sodium pyruvate and glutamine is recommended see 1 For the XF Glycolysis Stress Te...

Page 11: ...110 g Addition of sodium pyruvate from 100 mM liquid Final pyruvate concentration mL of pyruvate solution per 100 mL 0 5 mM 0 5 mL 1 0 mM 1 mL 2 0 mM 2 mL 4 Add the desired amount of L glutamine usin...

Page 12: ...nologies 25030 081 0 2 M Sterile Filter pH Meter 1N NaOH Method 1 Warm 100 mL of XF Base Medium to 37 C 2 Add the desired amount of L glutamine according to table below Final glutamine concentration m...

Page 13: ...ope to a Confirm cell health morphology seeding uniformity and purity no contamination b Ensure cells are adhered and no gaps are present c Make sure no cells were plated in the background correction...

Page 14: ...main in the utility plate and be placed flat on the work surface throughout the loading procedure Do not lift or angle the plate cartridge away from the work surface while loading 5 Handle the XF24 ca...

Page 15: ...bles b NOTE Do NOT tap any portion of the cartridge in an attempt to alleviate air bubbles This may cause compound leakage through the injection port Step 4 Inspect Injection Port Inspect complete loa...

Page 16: ...e sure the water bottle and clean bottle are filled with DI water and 70 ethanol respectively If the yellow light in front of the prep station is turned on it indicates that the volume is too low Fill...

Page 17: ...ote The Prep Station will prime the manifold with XF Assay Media and performs two aspirations and dispenses cycles There will be a pause in the operation then the prep station will perform a third asp...

Page 18: ...containing DI water from 1 3 Click Prime Manifold from the tab of the prep station software 4 Select Media 5 Click Start 6 After the cycle is finished transfer the tube to the 50 mL conical tube conta...

Page 19: ...or the purposes of providing richer data for discussion we will run the XF Glycolysis Stress Test and inject A Glucose B Oligomycin and C 2 Deoxy D glucose 2 DG In this experiment we will use 1 0 M ol...

Page 20: ...e hour prior to the assay 8 Prepare the stock compounds from the XF Glycolysis Stress Test For more details refer to the XF Glycolysis Stress Test User Guide a Important Use compounds the same day the...

Page 21: ...b Port B 1 M oligomycin final concentration in the well Load 62 L of the 10x solution into each Port B c Port C 50 mM 2 DG final concentration in the well Load 69 L of the 10x stock into each Port C 1...

Page 22: ...optimal concentration of oligomycin However for the purposes of providing richer data for discussion we will run the XF Cell Mito Stress Test and inject A oligomycin 4 concentrations B FCCP and C rote...

Page 23: ...a Important Use compounds the same day they are reconstituted Do not refreeze Discard any remaining compound b Remove foil pouch from XF Cell Mito Stress Test Kit box Each pouch contains reagents suff...

Page 24: ...0 5 M final concentration in the well iii Row C 1 0 M final concentration in the well iv Row D 2 0 M final concentration in the well b Port B 0 5 M FCCP final concentration in the well Load 62 L of ea...

Page 25: ...iding richer data for discussion we will run the XF Cell Mito Stress Test and inject A oligomycin optimal concentration B FCCP six different concentrations and C rotenone antimycin A Plate Layout FCCP...

Page 26: ...e same day they are reconstituted Do not refreeze Discard any remaining compound b Remove foil pouch from XF Cell Mito Stress Test Kit box Each pouch contains reagents sufficient for a completeXF Cell...

Page 27: ...Load 62 L of each 10x solution into the B ports in the appropriate columns shown below i Column 1 0 M final concentration in the well ii Column 2 0 125 M final concentration in the well iii Column 3...

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