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XF24 Training Manual
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Summary of Contents for Seahorse XF24
- Page 1 XF24 Training Manual...
- Page 2 XF24 FluxPak mini 101174-100 XF24 Islet Capture FluxPak XF Microplates # of Part # Description Microplates 100777-004 XF24 V7 PS Cell Culture Microplates 101122-100 XF24 Islet Capture Microplates XF Media & More Part # Description Quantity 100840-000 XF Calibrant 500ml bottle...
- Page 3 XF24 Recommended Assay Volumes XF MICROPLATE Recommended assay volume: 500-1,000 µL per well. When performing the medium exchange, ensure that the final volume of XF Assay Medium allows for injection volumes. The final volume in the wells during the assay must not exceed 1,000 µL.
- Page 4 Pre-training How does a Seahorse XF Analyzer work? Take 15 minutes to watch these six videos and you’ll have a good foundation for understanding XF Assays. Introductory videos available at http://www.seahorsebio.com/learning/videos.php a. Energy Pathways (2:06 minutes) b. Transient Microchamber (2:12 minutes) c.
- Page 5 Basic Procedure XF Assay Roadmap After Your Assay After Your Assay Next experiments Data interpretation Day of Assay Day Prior to Assay Planning Your Assay Prepare and run assay • Hydrate cartridge • Prepare assay • Seed cells medium • Obtain materials •...
- Page 6 Seed cells and incubate overnight in growth medium Hydrate sensor cartridge overnight Day Before XF Assay Day of XF Assay Add compounds to reagent ports Change medium to bicarbonate-free low-buffered assay medium Calibrate sensors XF Assay Contact Seahorse Technical Support: www.seahorsebio.com/techsupport...
- Page 7 The following are the materials required to run an XF24 assay. • XF24 Analyzer with Controller • Non-CO Incubator (37°C) or XF Prep Station • XF24 FluxPak (Part # 100850-001 ) or XF24 FluxPak mini (Part # 100867-100) • pH Meter • Inverted Phase Contrast Microscope • Pipette •...
- Page 8 Basic Procedure Available Kits and Reagents The following are materials offered by Seahorse Bioscience to assist you in designing assays to examine cellular metabolism. Each kit and reagent is designed to address your specific experimental question. If you have any questions about which kits and reagents may be appropriate for your experiments, please visit our website http://www.seahorsebio.com/...
- Page 9 ). This procedure describes recommendations for seeding adherent cell types for use with the XF24 Analyzer. A two-step seeding process is recommended when seeding XF24 cell culture microplates. The two-step process produces a consistent and even monolayer of cells. 1. Harvest and re-suspend the cells to desired final concentration to seed in 100 μL of growth medium.
- Page 10 (1) XF Cell Mito Stress Test Kit, and (2) XF Glycolysis Stress Test Kit Seahorse recommends the use non-buffered assay medium for XF assays to ensure accurate, functional measurements of metabolic phenotypes in an ambient environment. The optimal assay media for the XF Cell Mito Stress Test and the XF Glycolysis Stress Test are different.
- Page 11 Basic Procedure Final glucose concentration Grams of glucose per 100 mL 2.5 mM 0.045 g 5.5 mM 0.10 g 10 mM 0.18 g 25 mM 0.45 g 3. Add the desired amount of sodium pyruvate using powder or liquid using one of the following tables.
- Page 12 For one tissue culture plate: if rinsing manually, 100 mL of assay medium is sufficient; if rinsing using the XF Prep Station 200 mL of assay medium is sufficient. Materials Source Cat # XF Base Medium Seahorse Bioscience 102353-100 L-Glutamine (200 mM) Life Technologies 25030-081 0.2 µM Sterile Filter...
- Page 13 Microplates This basic procedure describes replacing the growth medium with assay medium for adherent cells grown in XF24 cell culture microplates prior to being assayed using an XF24 Analyzer. 1. Warm the pre-made Assay Medium to 37 (See basic procedure - Assay Media Preparation).
- Page 14 Do not hold the cartridge and utility plate between your thumb and fingers. Avoid traveling with the cartridge. To mitigate the accidental discharge of compounds prior to starting the assay, the best practice is to hydrate the cartridge and load the injection ports adjacent to the XF24 Analyzer.
- Page 15 Basic Procedure Step 3 – Load the XF24 Cartridge Using a p200 or a multichannel pipette, load 75 µL of injection compound into the desired injection ports according to your plate map/groupings using the following technique: 1. Hold the tips at a 45° angle 2.
- Page 16 *The medium bottle is not sterile. Make sure to remove an aliquot of the assay medium for drug dilutions. 4. Remove the lid from the tissue culture plate containing the cells. 5. Place the cells vertically (A1 facing the Seahorse logo head).
- Page 17 6. Launch the Seahorse Prep Station software by clicking on the “Seahorse XF Prep Station” icon 7. Select “Seahorse Guest.” 8. Click on the “Media Change” tab 9. Select “Do Prime” and type in the desired final volume in µL, for example 180 µL if using the XF96 or XFe96 Analzyer or 500 µL if using the XF24 or XFe24 Analyzer.
- Page 18 Cleaning the Prep Station 1. Fill up two 50 mL conical tubes with DI water. Fill up one 50 mL conical tube with 70% ethanol. 2. Remove the cap containing the tube from the assay medium bottle and place the tube into one of the 50 mL conical tube containing DI water from #1.
- Page 19 Basic Procedure Cell Density with the XF Glycolysis Stress Test The XF Glycolysis Stress Test is run with four different cell densities to determine the optimal cell density to use in your XF assays. In a typical cell density optimization assay, only three basal measurements need to be taken to determine the optimal cell seeding density.
- Page 20 Gently pipette up and down (~10 times) to solubilize the compounds. Vortex the 2-DG for at least 1 minute to ensure that it goes into solution. Volume of Assay Medium Final Concentration Glucose 3000 µL 100 mM Oligomycin 720 µL 100 µM 2-DG 3000 µL 500 mM Contact Seahorse Technical Support: www.seahorsebio.com/techsupport...
- Page 21 3 rate measurements after each injection. 12. On the Run Screen, Press Start and load the cartridge. 13. When prompted by the software, replace the Utility Plate with the Cell plate. Press Continue. Contact Seahorse Technical Support: www.seahorsebio.com/techsupport...
- Page 22 Row C: 1.0 µM final concentration in the well Row D: 2.0 µM final concentration in the well Port B: FCCP – 0.5 final concentration in the well µM Port C: rotenone/antimycin A – 0.5 final concentration in the well µM Contact Seahorse Technical Support: www.seahorsebio.com/techsupport...
- Page 23 Prepare serial dilutions of oligomycin in assay medium, as detailed below. Port A Tube [Final well] Stock Medium Oligomycin (µM) volume volume (µL) (µL) 2400 1500 1500 from a 1000 1000 from b 2000 Contact Seahorse Technical Support: www.seahorsebio.com/techsupport...
- Page 24 – 2 min – 3 min. Usually 3 basal rate measurements are taken prior to the first injection; then 3 rate measurements after each injection. 11. On the Run Screen, Press Start and load the cartridge. 12. When prompted by the software, replace the Utility Plate with the Cell plate. Press Continue. Contact Seahorse Technical Support: www.seahorsebio.com/techsupport...
- Page 25 Column 4: 0.50 µM final concentration in the well Column 5: 1.0 µM final concentration in the well Column 6: 2.0 µM final concentration in the well Port C: rotenone/antimycin A: 0.5 µM final concentration in the well Contact Seahorse Technical Support: www.seahorsebio.com/techsupport...
- Page 26 Prepare 3 mL of oligomycin in assay medium to achieve the desired final concentration determined in Training experiment 1 (or use 1 µM, which works for most cell types). Port A [Final well] Stock Medium Oligomycin (µM) volume volume (µL) (µL) 2,850 2,700 2,400 Contact Seahorse Technical Support: www.seahorsebio.com/techsupport...
- Page 27 – 2 min – 3 min. Usually 3 basal rate measurements are taken prior to the first injection; then 3 rate measurements after each injection. 11. On the Run Screen, Press Start and load the cartridge. 12. When prompted by the software, replace the Utility Plate with the Cell plate. Press Continue. Contact Seahorse Technical Support: www.seahorsebio.com/techsupport...
