liquid perfusion without the overhead · liquid perfusion without the overhead: fast, serial liquid...

Liquid Perfusion without the Overhead:Fast, Serial Liquid Replacement for PAM Assay Development

Ali Yehia, PhD!Director of IonFlux Product Development Fluxion Biosciences

© 2014 Fluxion Biosciences Inc.

Background

• Founded in 2005• Headquarters in California• Worldwide distribution• Winner of 5 NIH grants• Two time R&D 100 Award winner

• BioFlux (2008)• IonFlux (2010)

IonFlux BioFlux IsoFlux


IonFlux System TM

Built by Fluxion Biosciences, distributed and supported by Molecular Devices www.moldev.com


IonFlux 16 & HT

• Automated Patch Clamp System based on a unique in-plate liquid handling strategy

• No liquid handler so footprint akin to a simple plate reader

• Comes in two models: • IonFlux 16:

• 16 Amplifiers • IonFlux HT:

• 64 Amplifiers


Recording Plates• Standard SBS plate format

• 96 Wells for IF 16 • 384 Wells for IF HT

• Bottom of plate is replaced by intricate laminar flow system → providing an integrated

compound delivery and recording


• Two Traps: → Two independent recordings

• 8 different compounds or concentrations

• Two different IC per plate • Independent conduit:

• No mixing • No cross contamination • No pipetting delays • No electrode/compound

contact

Recording Plates


Cover of Assay for August 2012


System Integration


Mechanism of Action• Plates are primed with all solutions

get close to the action • Cells introduced and trapped • Seals are formed • Extra suction forms Whole Cell

Patch


Recording• Fast & successive compound

release • Continuous compound washing

within the gaps • No pipetting for compound

delivery allowing simultaneous application and recording across the plate

• 32 experiments completed in matter of minutes

• 500 compounds with 8 point dose response completed in few hours


Ensemble & Single Cell recording

• Ensemble recordings designed with 20 cells (High success rate) • Currents summed not averaged

• Single cell recording designed for highest quality (Giga-seals)

20-cell ensemble Single cell


Trapping Cells

• Transparent plates • Cells trapped and patched to

the side → Imaging possible

• Cell flow: • Increases access of cells

to patch holes • Can be interrupted to

trapping

© 2014 Fluxion Biosciences Inc.© 2012 Fluxion Biosciences. All rights reserved.

PneumaticPressure

InterfaceGasket

Electrode

Heating element

Temperature Control• Heating elements right under the

plate • Accurate temperature control with

positive feedback • Rapid changing of temperature

(~1 min)


Temperature Control


Flexibility in Solution Replacement

• IonFlux software provides extensive control over the delivery of compounds and solutions

• Wash steps are as simple as separating two compound additions

• Unique successive compound additions with no break in between

• Rapid exchange of solutions via parallel compound delivery system


Allosteric Modulators

• Both PAM and Agonist are needed to initiate response.

• The effect is not seen without agonist.

• In order to study the effect we need:

• Response consistency from agonist activation → Repeatability

• Flexibility and speed in solution displacement

Dawson GR et al 2005


GABAA Receptors

• Superimposed GABAA receptors current sweeps (HEK cells expressing α1β3γ2 - Eurofins PrecisION Cat# CYL3053)

• EC50 from 40 recording wells with normalized currents showing a value of 3.32 ± 0.1 µM

© 2014 Fluxion Biosciences Inc.© 2014 Fluxion Biosciences Inc.

GABAA Receptors: Agonists

• Different agonists providing different responses

• Dose response showing consistent results for all three agonists

muscimol > GABA > isoguvacine


Repeatability

• Superimposed GABA responses to 10 µM show minimal sweep to sweep variation, providing perfect substrate for the study of modulators effects


Repeatability

Consistent and repeatable response to successive additions of GABA is vital for the study of PAM effects


Positive Allosteric Modulation

• Successive addition of diazepam shows a marked increase in the response to GABA.


PAM Dose Response

• Successive addition of increasing concentrations of diazepam, showing marked increase in to the base1µM GABA response.

• Successive addition of increasing concentrations of triazolam, showing similar increase in base1µM GABA response.


PAM Dose Response


Rapid Liquid Displacement AssaysRapid Liquid Displacement Assays


Rapid Liquid Displacement Assays

Increasing GABA concentration recorded within the same sweep.


Rapid Liquid Displacement AssaysOpen channel modulation assays: 1 µM GABA → 1 µM Diazepam


Rapid Liquid Displacement AssaysOpen channel modulation assays + blocker


Rapid Liquid Displacement AssaysNMDA double agonist: 30 µM Glycine


Development


IonFlux System - Summary• The IonFlux system provides a suitable

and extensive platform for PAM assay development with benefits including: • Continuous flow of all solutions • Integrated or in-plate liquid

handling system • Rapid and serial displacement of

compounds • Extensive control in solution

exchange • Fast assay completion with

parallel execution of compound additions.

liquid perfusion without the overhead · liquid perfusion without the overhead: fast, serial liquid...

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