1. O 2 O 2 O 2 O 2 O 2 O 2 + H + H + H + H + H H + O 2 O 2 O 2 Assay Optimization in XF e 24 and XF e 96 Analyzers

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

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

4. Good Cell Culture Practice Watch for morphology or growth changes - Age - Contamination Passage before confluence Ensure consistent media components - Lot test serum - Fresh reagents & kept dark Monitor incubators - Humidity - CO 2

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 Cells per well (thousands) 100 200 300 Cells per well (thousands)

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

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

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 Seeding density Confluent cells may be outside dynamic range Shorten measurement time Increase mixing time

11. Whats 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 37 o C 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 37 o C

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 37 o C No CO 2

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

16. Making Stock Compounds 525 ul175 ul Assay Medium in each well 750 ul250 ul 10X Compound C 75 ul 25 ul XF24XF96 675 ul225 ul 9X Compound B 75 ul 25 ul 200 ul600 ul 8X Compound A 75 ul 25 ul 825 ul275 ul 11X Compound D 75 ul 25 ul

17. Some Compounds Need to be Optimized

18. Whats the Optimal Compound Concentration?

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