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Assay Optimization in XFe24 and XFe96 Analyzers
Presentation Outline
Cell Culture and Seeding
Cell Density Optimization
XF Cell Stress Test Compound Titration
XF Assay Flow Chart
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
Prepare sensor cartridge
Hydrate sensor cartridge overnight
Calibrate sensors
Assay
Add compounds to reagent ports
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- CO2
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?
Example: Mouse embryonic cortical neurons at Day 1 in culture (no neurites) versus Day 7
100 200 300Cells per well (thousands)
100 200 300Cells per well (thousands)
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
100 L of cells
1-5 hrs incubation
150 L of medium
80 L of cells96W: 1-step seeding
24W: 2-step seeding
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
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
What’s the proper seeding density?
07-14-06 MCF-7 cell number titration
0
100
200
300
400
500
600
700
10 20 30 40 50
Seeding cell number (thousands)
OC
R (
pm
ole
s/m
in)
0
10
20
30
40
50
60
70
80
EC
AR
mp
H/m
inOCRECAR
04-11-06 TR138 cell number titration
0
50
100
150
200
250
300
10 20 30 40 50 60
Seeding cell number (thousands)
OC
R (
pm
ole
s/m
in)
0
10
20
30
40
50
60
70
80
EC
AR
(m
pH
/min
)
OCRECAR
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
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 GlutamaxLow 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 GlutamaxLow phenol red (3 mg/L)
The user adds substrates, such as:GlucosePyruvateGlutamine (or Glutamax)
pH 7.4 ± 0.1 at 37oC
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 hr37oCNo CO2
XF Assay Flow Chart
Prepare sensor cartridge
Hydrate sensor cartridge overnight
Add substrates to reagent ports
Making Stock Compounds
525 ul 175 ul
Assay Medium in each well
750 ul 250 ul
10X Compound C75 ul 25 ul
XF24 XF96
675 ul 225 ul
9X Compound B75 ul 25 ul
200 ul600 ul
8X Compound A75 ul 25 ul
825 ul 275 ul
11X Compound D75 ul 25 ul
Some Compounds Need to be Optimized
What’s the Optimal Compound Concentration?
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
Prepare sensor cartridge
Hydrate sensor cartridge overnight
Calibrate sensors
Assay
Add compounds to reagent ports
Real-time data acquisition and output