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Assay Optimization in XF e 24 and XF e 96 Analyzers

O. O. O. O. O. O. O. O. O. 2. 2. 2. 2. 2. 2. 2. 2. 2. +. H. +. H. +. H. +. H. +. +. H. H. Assay Optimization in XF e 24 and XF e 96 Analyzers. Presentation Outline. XF Assay Flow Chart. Cell Culture and Seeding. Cell Density O ptimization.

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Assay Optimization in XF e 24 and XF e 96 Analyzers

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  1. O O O O O O O O O 2 2 2 2 2 2 2 2 2 + H + H + H + H + + H H Assay Optimization in XFe24 and XFe96 Analyzers

  2. Presentation Outline XF Assay Flow Chart Cell Culture and Seeding Cell Density Optimization XF Cell Stress Test Compound Titration

  3. XF Assay Flow Chart Add compounds to reagent ports Prepare sensor cartridge Prepare cells in XF plate Seed cells and incubate overnight in growth medium Hydrate sensor cartridge overnight Change medium to bicarbonate-free low-buffered assay medium Assay Calibrate sensors

  4. Good Cell Culture Practice • Watch for morphology or growth changes • - Age • - Contamination • Passage before confluence • Ensureconsistent media components • - Lot test serum • - Fresh reagents & kept dark • Monitor incubators • - Humidity • - CO2

  5. Factors dependent on your cell model • When, how and how many to plate? • Cell line or primary cell? • Proliferating or differentiated? • Require surface treatment? • Biological and/or physiological requirements?

  6. Example: Mouse embryonic cortical neurons at Day 1 in culture (no neurites) versus Day 7 100 200 300 100 200 300 Cells per well (thousands) Cells per well (thousands)

  7. XF Assay Flow Chart • Key Factors in Cell Seeding • Single cell suspension is optimal • Consistency • Consider cell attachment • Avoid edge effect Prepare cells in XF plate Seed cells and incubate overnight in growth medium

  8. 24W: 2-step seeding 150 L of medium 100 L of cells 1-5 hrsincubation 96W: 1-step seeding 80 L of cells

  9. Cell Seeding Number Must be Optimized • Factors to consider: • Good signal – XF24: ~ 50 – 400 pmol/min OCR • – XF96: ~ 20 – 160 pmol/min OCR • Nice, consistent monolayer – not necessarily confluent • Small magnitude of error

  10. Example: Primary rat hepatocytes require confluency to maintain hepatic features Cell number titration • Confluent cells may be outside dynamic range • Shorten measurement time • Increase mixing time Confluent Seeding density

  11. What’s the proper seeding density?

  12. XF Assay Flow Chart Prepare cells in XF plate Seed cells and incubate overnight in growth medium Change medium to bicarbonate-free low-buffered assay medium

  13. Assay Medium Depends on Assay:Starting from XF Base Medium, add substrates dependent on assay Glycolysis Stress Test Assay Medium (DMEM with NO sodium bicarbonate) NO Glutamax Low phenol red (3 mg/L) The user adds substrates, such as: Glutamine pH 7.35 ± 0.05 at 37oC Cell Mito Stress Test Assay Medium (DMEM with NO sodium bicarbonate) NO Glutamax Low phenol red (3 mg/L) The user adds substrates, such as: Glucose Pyruvate Glutamine (or Glutamax) pH 7.4 ± 0.1 at 37oC

  14. XF Assay Flow Chart Prepare cells in XF plate Seed cells and incubate overnight in growth medium Change medium to bicarbonate-free low-buffered assay medium 1 hr 37oC No CO2

  15. XF Assay Flow Chart Add substrates to reagent ports Prepare sensor cartridge Hydrate sensor cartridge overnight

  16. Making Stock Compounds Assay Medium in each well XF24 XF96 525 ul 175 ul 25 ul 75 ul 8X Compound A 600 ul 200 ul 75 ul 25 ul 9X Compound B 675 ul 225 ul 25 ul 75 ul 10X Compound C 750 ul 250 ul 25 ul 75 ul 11X Compound D 825 ul 275 ul

  17. Some Compounds Need to be Optimized

  18. What’s the Optimal Compound Concentration?

  19. XF Assay Flow Chart Add compounds to reagent ports Prepare sensor cartridge Prepare cells in XF plate Seed cells and incubate overnight in growth medium Hydrate sensor cartridge overnight Change medium to bicarbonate-free low-buffered assay medium Assay Calibrate sensors Real-time data acquisition and output

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