The Application of Cell-Based Impedance Technology in Drug - - PowerPoint PPT Presentation

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The Application of Cell-Based Impedance Technology in Drug - - PowerPoint PPT Presentation

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The Application of Cell-Based Impedance Technology in Drug Discovery Yama A. Abassi, PhD Sr. Director Cell Biology and Assay Development ACEA Biosciences www.aceabio.com ACEA Biosciences Founded in early 2002 Located in San

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The Application of Cell-Based Impedance Technology in Drug Discovery

Yama A. Abassi, PhD

  • Sr. Director

Cell Biology and Assay Development ACEA Biosciences

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ACEA Biosciences

  • Founded in early 2002
  • Located in San Diego, CA
  • Mission: Integration of microelectronics with cell biology and molecular

biology for providing innovative and cost-effective microelectronic biological analysis systems and applications for life science industry and clinical diagnostics

  • ACEA’s first product was marketed under the brand name of RT-CES

system in 2004

  • In November of 2007 Roche and ACEA Biosciences entered into an

exclusive agreement for the development, supply and distribution for ACEA Bioscience’s real-time cell assay technology. Under the terms of the agreement, RAS will exclusively market systems for real-time cell analysis, based on ACEA Bioscience's impedance-based technology

  • The first joint Roche/ACEA product is marketed under the brand name
  • f xCELLigence
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xCELLigence RTCA SP System

Analyzer Computer and Software Plate Reader(RTCA SP) (in CO2 incubator) Gold Microelectrode Covers 80% of Well Area E-Plate

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Electronic Sensor Technology Applied to Cell Biology: Principle of Operation Electronic Sensor Technology Applied to Cell Biology: Principle of Operation

Derivati Derivation of Cell Index n of Cell Index

A dimensionless parameter termed Cell Index (CI) is derived as a relative change in measured electrical impedance to represent cell status. Several features of the CI are summarized: 1. When cells are not present or are not well-adhered on the electrodes, then the CI is zero 2. Under the same physiological conditions when more cells are attached on the electrodes, then the CI values are larger. Thus, CI is a quantitative measure of cell number present in a well. 3. Additionally, change in a cell status, such as cell morphology, cell adhesion or cell viability will lead to a change in CI.

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Advantages of xCELLigence System for Cell-Based Assays and Drug Discovery Applications

Label free, no reporters Non-invasive measurement Real-time Monitoring

  • Short-term (milliseconds)
  • Long-term (days and weeks)

Continuous QC

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Cell-based Assays:

Traditional Methods

Seed cells Treatment: e.g.: drug compound Traditional methods: Labeling (e.g. optical) & End-point Measurement Initiate Experiment Data Analysis 18-24 Hours 24-48 Hours 1-24 Hours

Black Box

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Cell-based Assays:

Traditional Methods vs xCELLigence System

Seed cells Treatment: e.g.: drug compound Traditional methods: Labeling (e.g. optical) & End-point Measurement Initiate Experiment Data Analysis

xCELLigence System :

  • Label-free: Electronics-based detection
  • Real-time: Continuous measurement, data analysis and display
  • Therefore, both short term and long term compound effects can be

captured

18-24 Hours 24-48 Hours 1-24 Hours

Continuous QC

1 2 3 4 5 6 7 10 20 30 40 50 60 70 Time (Hours) N

  • rm

a lize d C e ll In d e x Control Cmpd A Cmpd B Cmpd C Cmpd D

Treatment

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  • Cell Proliferation
  • Cell Quality
  • Compound-mediated Cytotoxicity
  • Cell-mediated Cytotoxicity
  • Cell Adhesion and Spreading
  • Functional Monitoring of Receptor Tyrosine Kinase Signaling
  • Functional Monitoring of GPCR Signaling
  • IgE Receptor Function
  • Cell Invasion and Migration
  • Barrier Function
  • Viral Cytopathogenecity

Applications Developed on the xCELLigence System

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  • Cell Proliferation
  • Cell Quality
  • Compound-mediated Cytotoxicity
  • Cell Response Profiling
  • Cell-mediated Cytotoxicity
  • Cell Adhesion and Spreading
  • Functional Monitoring of Receptor Tyrosine Kinase Signaling
  • Functional Monitoring of GPCR Signaling
  • IgE Receptor Function
  • Cell Invasion and Migration
  • Barrier Function
  • Viral Cytopathogenecity

Applications Developed on the xCELLigence System

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Time-Dependent Cell Response Profiling

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The Road to Cellular Cytotoxicity Takes Many Twists and Turns

10 20 30 40 50 60 70 80 1 2 3 4 5 DMSO 0.125 uM 0.25 uM 0.5 uM 1 uM 2 uM 4 uM 8 uM

Time (hours) Normalized Cell Index

1 2 3 4 5 6 7 20 40 60 80 100 120 Time (hours) Normalized CI 10 uM 1.1 uM 0.12 uM 0.0137 uM 0.0016 uM Ctrl

Tunicamycin

0.5 1 1.5 2 2.5 10 20 30 40 50 60 Time (hours)

N orm a lize d C I 10 uM" 3.3 uM 1.1 uM 0.33 uM Control

Proteasome Inhibitor N-Glycosylation Inhibitor Anti-Mitotic

0.5 1 1.5 2 2.5 3 3.5 20 40 60 80 100 Time (hours) Norm alized CI 10 uM 1 0.12 Ctrl

DNA Damaging

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Are Impedance-Based Cell Response Profiles Predictive of Biological Mechanism ?

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TCRP Approach

Seed 4000 A549 Cancer Cells in 96 well E-Plates Treat with Compounds at a final concentration Of 20 μM Monitor the cellular response for 48 hours Compare cytological profiles

O O OH OH O HO

E-Plate

Add Cells Add Compound

Real-Time Continuous Monitoring

Monitor 20-24 hours Monitor 48 hours

Impedance-based real-time cellular response profile

Interdigitated gold microelectrodes

Spectrum Compound Library from MS Discovery (Collection of FDA approved drugs, nature compounds Experimental compounds, insecticides and herbicides)

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TCRP from Screen Short-term response (w/in 1 hour) Long Term Response (1-48 hours) Hit criteria (25% of control) Hit criteria (25% and 40%)

Hit Selection and Clustering Analysis

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TCRP with Known Mechanisms

0.5 1 1.5 2 2.5 3 3.5 20 40 60 80 Time (hours) Normalized CI Ctrl Colchicine 1 2 3 4 5 20 40 60 80 Time (hours) Normalized CI Ctrl Etoposide 1 2 3 4 5 6 7 20 40 60 80 Time (hours) Normalized CI Ctrl Hydrocortisone 1 2 3 4 20 40 60 80 Time (hours) Normalized CI Ctrl Emetine

1 2 3 4 20 40 60 80 Time (hours) Normalized CI Ctrl Azelastine

1 2 3 4 5 20 40 60 80 Time (hours) Normalized CI Ctrl Methiothepin

1 2 3 4 20 40 60 80 Time (hours) Normalized CI Ctrl Amlodipine

1 2 3 4 5 6 7 20 40 60 80 100 Time (hours) Normalized CI Ctrl Trichostatin A

Short-Term Response Long-Term Response

Anti-mitotics Protein-Synthesis DNA Damaging Nuclear Hormone HDAC Inhibitors Anti-Histamine Serotonin Receptor Antagonist L-Type Voltage-gated Ca Channel Inhibitor

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TCRP with Known Mechanisms

0.5 1 1.5 2 2.5 3 3.5 20 40 60 80 Time (hours) Normalized CI Ctrl Colchicine 1 2 3 4 5 20 40 60 80 Time (hours) Normalized CI Ctrl Etoposide 1 2 3 4 5 6 7 20 40 60 80 Time (hours) Normalized CI Ctrl Hydrocortisone 1 2 3 4 20 40 60 80 Time (hours) Normalized CI Ctrl Emetine

1 2 3 4 20 40 60 80 Time (hours) Normalized CI Ctrl Azelastine

1 2 3 4 5 20 40 60 80 Time (hours) Normalized CI Ctrl Methiothepin

1 2 3 4 20 40 60 80 Time (hours) Normalized CI Ctrl Amlodipine

1 2 3 4 5 6 7 20 40 60 80 100 Time (hours) Normalized CI Ctrl Trichostatin A

Short-Term Response Long-Term Response

Anti-mitotics Protein-Synthesis DNA Damaging Nuclear Hormone HDAC Inhibitors Anti-Histamine Serotonin Receptor Antagonist L-Type Voltage-gated Ca Channel Inhibitor

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TCRP of Anti-mitotic Compounds

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Ctrl 6 h (Phase I) 14 h (Phase II) 24 h (Phase III) 48 h (Phase IV)

10 20 30 40 50 60 70 80 90 20 40 60 80 Time (h) Mitotic Index (%) Untreated 12.5 nM Paclitaxel

Mitotic Index

Characterization of Impedance- Based Anti-mitotic Profile

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Validation of Mitotic Arrest Profile

1 2 3 4 5 6 7 20 40 60 80 100 Time (h) Normalized CI S-Trityl-L-Cysteine DMSO

Eg5 Small Molecule Inhibitor

anti- tubulin Ab anti- PH3

Ctrl

S-Trityl-L-Cysteine

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Compounds with Anti-mitotic Profile from the Spectrum Collection

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Systematic Analysis of Impedance-Based Cell Response Profiles

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Curve Classification Algorithm and Display

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CELECOXIB TAMOXIFEN FLUPHENAZINE PERPHENAZINE METHIOTHEPIN COLCHICINE NOCODAZOLE NOSCAPINE beta-PELTATIN ESTRADIOL PUROMYCIN PYRROMYCIN CYCLOHEXIMIDE EMETINE LYCORINE TENIPOSIDE CAMPTOTHECIN STROPHANTHIDIN OLEANDRIN ETOPOSIDE BUDESONIDE HOMATROPINE HYDRALAZINE HYDROCORTISONE METHYLPREDNISOLONE Ca Modulator Anti-mitotic Protein Synthesis In DNA Damaging Nuclear Receptor

Clustering Analysis

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www.aceabio.com Valdecoxib

1 2 3 4 5 20 40 60 80 Time (hours) Normalized CI 20 uM 10 uM 5 uM Ctrl

Rofecoxib

1 2 3 4 5 20 40 60 80 Time (hours) Normalized CI 20 uM 10 uM 5 uM 2.5 uM

Deracoxib

1 2 3 4 5 20 40 60 80 Time (hours) Normalized CI 20 uM 10 uM 5 uM 2.5 uM

N N S O O NH2 F F F O N N S O O NH2 F F F S N O O O H

2N

S O O O O

TCRP COX-2 Inhibitors

COX-2 Inhibitors

Valdecoxib Rofecoxib Celecoxib Deracoxib

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1 2 3 4 5 20 40 60 80 Time (hours) Normalized CI 20 uM 10 uM 5 uM Ctrl

Celecoxib

1 2 3 4 5 20 40 60 80 Time (hours) Normalized CI 20 uM 10 uM 5 uM 2.5 uM

Rofecoxib

1 2 3 4 5 20 40 60 80 Time (hours) Normalized CI 20 uM 10 uM 5 uM 2.5 uM

Deracoxib

1 2 3 4 5 20 40 60 80 Time (hours) Normalized CI 20 uM 10 uM 5 uM 2.5 uM

N N S O O NH2 F F F O N N S O O NH2 F F F S N O O O H

2N

S O O O O

COX-2 Inhibitors

Valdecoxib Rofecoxib Celecoxib Deracoxib

TCRP COX-2 Inhibitors

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0.2 0.4 0.6 0.8 1 1.2 26.7 26.8 26.9 27 27.1 27.2 27.3 Time (hours) Normalized CI 10 uM BAPTA AM DMSO Untreated

BAPTA AM 50 uM Celecoxib

Validation of Celecoxib as Modulator of Intracellular Calcium Levels

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0.2 0.4 0.6 0.8 1 1.2 26.7 26.8 26.9 27 27.1 27.2 27.3 Time (hours) Normalized CI 10 uM BAPTA AM DMSO Untreated

BAPTA AM 50 uM Celecoxib

  • 100

100 200 300 400 500 600 Mean Fluorescence (Arbitrary Units)

T h a p s i g a r g i n 50 uM 25 uM 12.5 uM 6.25 uM

Celecoxib

Validation of Celecoxib as Modulator of Intracellular Calcium Levels

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Advantages of Short Term and Long Term Monitoring of Cellular Response Profiles

H N N H S O O CH3 CH3 OH

Monastrol

1 2 3 4 5 10 20 30 40 50 60 Time (hours) Normalized CI Ctrl Monastrol

HeLa Cells

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Advantages of Short Term and Long Term Monitoring of Cellular Response Profiles

H N N H S O O CH3 CH3 OH

Monastrol

1 2 3 4 5 10 20 30 40 50 60 Time (hours) Normalized CI Ctrl Monastrol

HeLa Cells

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Advantages of Short Term and Long Term Monitoring of Cellular Response Profiles

H N N H S O O CH3 CH3 OH

Monastrol

1 2 3 4 5 10 20 30 40 50 60 Time (hours) Normalized CI Ctrl Monastrol

HeLa Cells

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Advantages of Short Term and Long Term Monitoring of Cellular Response Profiles

H N N H S O O CH3 CH3 OH

Monastrol

1 2 3 4 5 10 20 30 40 50 60 Time (hours) Normalized CI Ctrl Monastrol

HeLa Cells

Ctrl Monastrol Phospho-Histone H3

Long Term

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Advantages of Short Term and Long Term Monitoring of Cellular Response Profiles

H N N H S O O CH3 CH3 OH

Monastrol

1 2 3 4 5 10 20 30 40 50 60 Time (hours) Normalized CI Ctrl Monastrol

HeLa Cells

Ctrl Monastrol Phospho-Histone H3

Long Term

N H H3C H

3COOC

CH3 NO2 O

nifedipine

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Advantages of Short Term and Long Term Monitoring of Cellular Response Profiles

H N N H S O O CH3 CH3 OH

Monastrol

1 2 3 4 5 10 20 30 40 50 60 Time (hours) Normalized CI Ctrl Monastrol

HeLa Cells

400 800 1200 1600 2000 20 40 60 80 100

Monastrol (uM) Mean Relative Fluorescence

Ctrl Monastrol

Voltage-Gated Calcium Level Modulation

Phospho-Histone H3

Short Term Long Term

N H H3C H

3COOC

CH3 NO2 O

nifedipine

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Functional Monitoring of GPCR Signaling

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GPCR Activation Leads to Modulation

  • f the Actin Cytoskeleton
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0.8 1 1.2 1.4 1.6 1.8 2 6 8 10 12 14 16 18 Time (h) Normalized CI

Histamine CTR

0.7 0.9 1.1 1.3 1.5 7 9 11 13

Time (h) Normalized CI Vasopressin CTR

Phalloidin anti-Paxillin Unstimulated + Histamine Phalloidin anti-Paxillin Unstimulated + Vasopressin

Dynamic Monitoring of GPCR-mediated Morphological Dynamics Using the xCELLigence RTCA System

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Functional Monitoring of Gq Coupled Receptors on the xCELLigence RT-CA System

  • 14 -13 -12 -11 -10 -9
  • 8
  • 7
  • 6
  • 5
  • 4
  • 3

1500 2000 2500 3000 3500 4000

EC50= 19 nM

[Histamine] Log M [3H]IP3 bound (cpm)

  • 13 -12 -11 -10
  • 9
  • 8
  • 7
  • 6
  • 5
  • 4

0.8 1.0 1.2 1.4 1.6 1.8 2.0

EC50=1.7 nM

[Histamine] Log M Normalized CI 0.8 1 1.2 1.4 1.6 1.8 2 7 9 11 13 15 17 Time (hr) Normalized CI

His 30 uM His 30 nM His 30 pM CTR

Histamine H1 Receptor RT-CA Assay 3H-IP3 Assay

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Functional Monitoring of Gs Coupled Receptors on the xCELLigence RT-CA System

0.8 1 1.2 1.4 12 14 16 18 Time (hr) Normalized CI

SKF 38393, 100 uM SKF 38393, 1 nM SKF 38393, 0.1 nM CTR

  • 12 -11
  • 10
  • 9
  • 8
  • 7
  • 6
  • 5
  • 4
  • 3

1.10 1.15 1.20 1.25 1.30 1.35 1.40

EC50 = 1.7 nM

[SKF 38393] Log M normalized CI

  • 12 -11 -10
  • 9
  • 8
  • 7
  • 6
  • 5
  • 4
  • 3

10 20 30 40 50 60 70

EC50= 1.9 nM

[SKF 38393] Log M [cAMP] pmoles/well

Dopamine1 Receptor RT-CA Assay cAMP Assay

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Functional Monitoring of Gi Coupled Receptors on the xCELLigence RT-CA System

0.8 1 1.2 1.4 1.6 1.8 12 14 16 18 Time (hr) N orm alized C I

8-OH-DPAT, 100 uM 8-OH-DPAT, 10 nM 8-OH-DPAT, 0.1 nM CTR

  • 12 -11 -10
  • 9
  • 8
  • 7
  • 6
  • 5
  • 4
  • 3

1.0 1.1 1.2 1.3 1.4

EC50 = 19 nM

[8-OH DPAT] Log M Normalized CI

  • 14 -13 -12 -11 -10 -9
  • 8
  • 7
  • 6
  • 5
  • 4

5 10 15 20

EC50 = 6 nM

[8-OH-DPAT] Log M [cAMP], nmol/well

5-HT1A Receptor RT-CA Assay cAMP Assay

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0.8 1 1.2 1.4 1.6 1.8 16 18 20 22 Time (hr) Normalized CI

Histamine 100 uM Histamine 160 nM Histamine 30 nM CTR

Histamine Receptor (Gq) in HeLa Cells

1 1.2 1.4 1.6 1.8 10 12 14 16 18 Time (hr) No rmalized CI Calcitonin 5 uM Calcitonin 20 nM Calcitonin 1 pM CTR

Calcitonin Receptor (Gs) in CHO Cells

0.9 1 1.1 1.2 1.3 22 23 24 Time (hr) Normalized CI DPDPE 30 uM DPDPE 3 nM DPDPE 30 pM CTR

Opioid Receptor (Gi) in NIE115 Cells

  • 12
  • 11
  • 10
  • 9
  • 8
  • 7
  • 6
  • 5
  • 4

0.95 1.00 1.05 1.10 1.15 1.20 1.25

EC50 = 2 nM

[DPDPE] Log M Normalized CI

  • 14 -13 -12 -11 -10
  • 9
  • 8
  • 7
  • 6
  • 5

1.0 1.1 1.2 1.3 1.4 1.5

EC50 = 385 pM

[Calcitonin] Log M Normalized CI

  • 9
  • 8
  • 7
  • 6
  • 5
  • 4
  • 3

1.0 1.2 1.4 1.6 1.8

EC50= 146nM

[Histamine] Log M Normalized CI

Dynamic Monitoring of Endogenous Receptors Using the xCELLigence System

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Dynamic Monitoring of Receptors in Disease-Relevant Cell Types

0.2 0.4 0.6 0.8 1 1.2 1.4 65 70 75 80 85 90 Time (Hours) Normalized CI 750 nM 150 nM 30 nM 6 nM 1.2 nM 0.3 nM Ctrl

Cor.AT Cells from Axiogenesis Mouse ES Cell-Derived Cardiomyocytes 100% Pure Population

EC- EC-50= 3. 50= 3.1 n 1 nM β2 Ad 2 Adrene renergic Recep rgic Recepto tor Activation in Activation in Cardiomyocyte- Cardiomyocyte-Di Differentiated fferentiated Co Cor.AT r.AT Cells Cells

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Identification of Histamine H1 Receptor Inverse Agonist using the xCELLigence RT-CA System

0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 10 12 14 16 18 20 Time (h) Normalized CI Histamine 20 nM Loratidine 100 uM 0.8 1 1.2 1.4 1.6 1.8 2 6 8 10 12 14 16 18 Time (h) Normarlized CI Histamine 100 nM CTR

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List of Receptors Functionally Monitored by the xCELLigence System

  • GPCR
  • RTK
  • FcR (IgE and IgG)
  • TCR
  • Death Receptors (FasR, TNFR)
  • Integrins
  • Toll Receptors
  • Nuclear Hormone Receptors
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Summary

  • Impedance-based monitoring of cellular status using the

xCELLigence platform allows for monitoring of both short term and long term responses

  • The ability to monitor short and long term responses within the

same experiment provides cytological profiles which can be predictive of mechanism of action

  • The non-invasive nature of impedance readout provides the

advantage of working with primary cells or disease relevant cells both for long term cytotoxicity studies and short term receptor responses