FLEXstation - Molecular Devices

FLEXstation• Xenon lamp and PMT– 250-850nm• incidental optics– improved sensitivity• Integrated 8-channel robot– real-time kinetic data• Capable of ratiometric anddual dye measurement• On-line data analysis(Softmax Pro)• Limited throughput

Membrane potentialMethodology• HEK293 cells expressing voltage sensitive K +channel• Cells washed with Hank’s buffer (3x100ul)• 100ul FLIPR Membrane Potential Assay Reagentadded to cells• Cells incubated for 1hr, 37°C• Fluorescence monitored in FLEXstation/FLIPRafter addition of agonist

FLIPRFLEXMembrane potential - Riluzole-inducedhyperpolarisationFluorescence IntensityFluorescence Intensity12000100008000450003500025000150000 25 50 75 100 125Time(s)[Riluzole] (µM)10005003002001005030200 25 50 75 100 125Time(s)

GPCR - [Ca 2+ ] irelease from intracelluar storesMethodology• Orexin-1 (OX 1), G q-linked GPCR involved in feedingand the architecture of sleep• CHO cells expressing Orexin-1 or Orexin-2 receptors• Loaded with Fluo-4 (4µM), 1hr, 37°C• Washed 4x in Tyrodes buffer• Antagonists/buffer control incubated for 30min, 37°C• Orexin-A or Orexin-B added using FLEXstation

GPCR - [Ca 2+ ] irelease from intracelluar storesFluorescence intensity3000025000200001500010000[Orexin-A] (nM)0.313103010030010000 10 20 30 40 50 60 70Time(s)

MethodologyIon Channel - extracellular Ca 2+ influx• Vanilloid receptor (VR1), non-specific cationchannel involved in nociception• Stimulated by pungent plant extract capsaicin• 1321-N1 cells expressing VR1 loaded with Fluo-4,2hrs, 25°C• Washed 4x in Tyrodes buffer• Antagonists/buffer control incubated for 30min, 25°C• Agonists added using FLEXstation/FLIPR

Ion Channel - extracellular Ca 2+ influxFLEXFLIPR60000150Increase in fluorescence4000020000% Capsaicin maximum100500-10 -9 -8 -7 -6 -5 -4log[Agonist] (M)CapsaicinRTXOlvanilPPAHV0-10 -9 -8 -7 -6 -5 -4log[Agonist] (M)

Ion Channel - extracellular Ca 2+ influx15000Increase in fluorescnce1000050000-10 -9 -8 -7 -6 -5 -4log [Capsazepine] (M)

Ion Channel - extracellular Ca 2+ influxIncrease in fluorescence2000015000100005000Row1234567891011120-9 -8 -7 -6 -5 -4log [Capsaicin] (M)

Ratiometric imaging using the FLEXstation•Dual excitation dyes– e.g. Fura-2– On binding Ca 2+ theexcitationwavelength shifts– ratio intensity at twowavelengths

Advantages of ratiometric imaging• Signal is not dependent on– dye concentration– illumination intensity– optical path length• Effects of spatial variation and dye loss areminimised and a more stable signal obtained• Increased sensitivity• Absolute changes in calcium concentrations canbe calcuated

Ratiometric imaging with Fura-212000∆ Fluorescence800040000-4000λ ex (nm)380 340[Capsaicin]10µM300nMControl-8000-120000 20 40 60 80Time(s)

Ratiometric imaging with Fura-2Fura-2Fluo-41.560000FI 340nm/FI 380nm1.00.5Increase in fluorescence40000200000.0-10 -9 -8 -7 -6 -5 -4log [Agonist] (M)CapsaicinRTXOlvanilPPAHV0-10 -9 -8 -7 -6 -5 -4log[Agonist] (M)

Summary• Demonstrated the utility of the FLEXstation for theinvestigation of-GPCRs- ion channels e.g. Ca2+ influxmembrane potential• Pharmacology using FLEXstation compares wellwith that using FLIPR• Demonstrated the increased functionality of theFLEXstaion e.g. use of dyes NOT possible inFLIPR

AcknowledgementsGlaxoSmithKlineDarren SmartIzzy BoyfieldMartyn WoodStephen BroughJennie HeathFrances JewittMolecular DevicesSimon LydfordSean Tyacke