Section9:Impedance-Based Technologies

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Impedance-Based Technologies

Introduction

Impedance-based assay systems for cell-based assays measure changes in electrical impedance relative to a voltage applied to a cell monolayer. They allow measurement of activation of all receptor types including G protein-coupled receptors, tyrosine kinase receptors, and some nuclear receptors. Upon activation of cell surface receptors, signal transduction pathways are initiated causing cellular morphological changes. Production of intracellular effectors results in changes in the cellular cytoskeleton which are reflected in changes in the flow of current across and between the cells in the monolayer. This change in the flow of current around and through cells is represented in a single well by an overall change in the impedance within that well (Figure 1). Impedance-based assay technologies are universal and require no labels or special reagents. They may be used with either transfected or endogenous receptors and for primary cells.

Two example impedance-based assay platforms are the RT-CES® from Acea Biosciences and the CellKey™ System from MDS Analytical Technologies.

Overview of RT-CES®

ACEA Biosciences RT-CES®: Cells are seeded into the ACEA RT-CES® 96-well plate which contains electrodes in the bottom of each well. After monitoring cell impedance through an equilibration period, the plate is removed from the instrument and compound additions are made, either manually or with external liquid handling instrumentation. The plate is returned to the incubator and the connection with the instrument is re-established. Monitoring of impedance continues. Responses are reported as “Cell Index”, a parameter derived from impedance measurements.

Features:

• System is set up in an incubator, so cells may be continuously monitored over long periods (eg. for growth curves and proliferation assays).
• With optional RT-CIM® module, cell invasion and migration assays may be performed.

Overview of CellKey™ System

MDS Analytical Technologies CellKey™ System: The CellKeyTM System is based on a label-free technology called Cellular Dielectric Spectroscopy (CDS), which is capable of measuring complex impedance changes in cell monolayers. Impedance (Z) is related to the ratio of voltage to current (Z=V/I) as described by Ohm’s law. Cells are seeded onto a CellKeyTM microplate that contains electrodes at the bottom of each well. The CellKeyTM instrument applies voltage across the electrodes producing electrical currents that flow around and between cells (extracellular current, iec) and through cells (transcellular current, itc) (Figure 2A).

The CellKey™ System measures changes in impedance that occur in each well upon stimulation of cell surface receptors. Contributors to the impedance as measured in each well are changes in cell adherence to substrate, changes in cell shape and volume, and changes in cell-cell interactions. These dynamic cellular changes affect the flow of extracellular and transcellular current and hence the magnitude and characteristics of the impedance measured. In practice, the extracellular current is what contributes most of the signal.

Changes in impedance are captured in real time and are quantified. In addition, the unique response profiles produced after receptor activation are indicative of the G-protein coupling type (Figure 2B). Applications of this technology include receptor panning, signal pathway identification and deconvolution, hit identification, enhanced selectivity screening, and pharmacological profiling for potency and efficacy.

Features:

• Built in liquid handling allows continuous monitoring of responses from the moment of compound addition.
• May be integrated with lab automation.
• Both adherent and suspension cells may be used as well as primary cells.
• Assays may be run at either 37oC or room temperature.
• Response profiles have been correlated to G protein-coupling status.
• Small sample 96-well plates are available with 1/20 the well surface area as the standard 96-well plate, allowing a 75% or greater reduction in cells required as well as savings on test compounds.
• Easy to use software for data analysis.

Sample Protocol for CellKey™ (MDS Analytical Technologies)

Also see CellKey™ Assay Development Guide provided by MDS Analytical Technologies and the CellKey™ Operator’s Manual.

1. Assay Buffer: Hank’s Balanced Salt Solution (HBSS) with Ca and Mg, 20 mM Hepes, 0.1 % BSA. If BSA is included, it should be fatty acid-free (Sigma #A0281 or #A6003) to avoid activation of endogenous fatty acid receptors on cells. (Other buffers such as Tyrodes, PBS, or culture media may also be used.)
2. Harvest, count, and resuspend cells to yield the appropriate density determined during assay optimization. Cells should be just confluent at the time of assay. Seed cell plate with 150 µl/well.
   • Adherent cells are usually plated the day before the assay in culture medium and incubated at 37oC, 5% CO2 overnight. The CellKey™ 96-well microplates may be treated with surface coatings such as collagen or poly-d-lysine to improve adherence if desired.
   • Suspension cells are harvested, washed three times in assay buffer, counted and resuspended to the appropriate concentration in assay buffer. 135 μl of cell suspension is dispensed into each well. Cells are allowed to settle for a minimum of 30 minutes at room temperature before assay.
3. Prepare 10X agonist and antagonist compounds in assay buffer in 96-well plates.
4. Load cell plate, pipet tips, compound plate, and reservoir with assay buffer into the CellKey™ System.
5. Default protocols are available for fluid exchange and stimulation/data acquisition, depending on whether cells are adherent or suspension, or if agonist or antagonist responses are to be measured. These protocols may be edited or used as written. These protocols control the fluidic parameters (tip height, volumes, speed, etc) as well as assay temperature.
6. Stimulation and data acquisition may be performed at either 37oC or room temperature.
7. Fluid exchange is performed on adherent cells plated in culture media. It may be done using the CellKey™ System or off-line with other liquid handling instrumentation. It consists of aspiration of culture media, washing the wells 3 times with assay buffer, and leaving 135 μl of assay buffer in the wells. There is no need to perform fluid exchange on suspension cells.
8. Generally, cells are equilibrated for 20 minutes after fluid exchange. If antagonists are to be added, they are pipetted by the CellKey™ System immediately after fluid exchange so pre-incubation can proceed during the equilibration period. 15 μl of 10X antagonist is added to 135 μl well volume (Figure 3).
9. The agonist stimulation protocol is initiated after equilibration. Generally 15 μl of 10X agonist is added to 135 μl in the plate. If an antagonist has been added previously, well volume = 150 μl, and 16.5 μl of 10X agonist is added.
10. For data collection, default settings in the CellKey™ System software are provided for most adherent cells (for example HEK293 and CHO). Details are found in the Operator’s Manual. Response time is usually 5-15 minutes following a 2 minute baseline data collection. Quantifying responses is typically done by calculation of the maximum change from baseline.
11. In addition to quantifying the response, qualitative MOA information may be obtained by examination of kinetic response profiles.

CellKey™ System Results and Data Analysis

1. Kinetic Responses for a selected individual well or all wells are displayed on the screen. dZiec (extracellular current) responses are quantified by the instrument software (Figure 4) by either, subtraction of the minimum impedance reading from the maximum impedance reading, the maximum impedance reading from baseline, or the impedance reading at a specified point in time during the stimulation period. Data is exported to Excel and saved.
   Figure 4A. Kinetic profiles from 96-well plate

   
   

   
   Figure 4B. Kinetic Profile from individual well

   
   

2. Max – min dZiec responses can be plotted vs compound concentrations to generate concentration response curves and calculate EC50 / IC50. (Please refer to Section XI, D, Data Standardization.)
   Figure 5. Example: Agonist concentration response curve EC50 for agonist stimulated increase in impedance for a Gq coupled GPCR correlates with the EC50 for IP-1 accumulation using IP-One HTRF®.

   
   

   
   Figure 6. Example: Antagonist concentration response curve

Helpful Hints for performing CellKey™ Assays

• Allowing cell plates to rest for at least 15 minutes before placing in the incubator for overnight incubation allows cells to settle evenly to the bottom of the well and improves variability.
• Evaporation control: Media evaporation during overnight incubation can lead to “edge effects” due to effects on cell growth. Use of MicroClime™ Environmental Lids (Labcyte Inc. #LLS-0300) or BREATHseal™ (Greiner Bio-One #676051) is recommended on cell plates to reduce evaporation.
• Changes in buffer components such as DMSO, BSA and cations, can lead to responses in cells reflected by changes in impedence. Buffer constituents should be consistent between the cells in the microplate and compounds added. If the final DMSO concentration will be higher than 0.1%, it is necessary to include the same concentration of DMSO in the equilibration buffer.
• In order to avoid changes in compound concentration due to evaporation before or during incubation in the instrument, a pre-scored plate seal such as the EZ Pierce Plate Sealing films (Sigma) should be used on the compound plates.
• Make sure compound plate is warmed to at least room temperature before running the assay to avoid temperature differences between cell plate and compounds.
• For characterization of receptor-mediated response profiles, best results are obtained at 28oC rather than 37oC. This may be due to slower kinetics at the lower temperature.
• CellKey™ Small Sample 96-well plates are available that have the footprint of a 96-well plate but the well area of a 1536-well plate. This allows a reduction of at least 75% in cells required, and also reduces volume of compound needed. CellKey™ System Technical Note 1 details special considerations when using these plates.
• CellKey™ System microplates may be coated with collagen (Sigma C9791) or poly-d-lysine (Sigma P6407) or any other coating material of interest. If using poly-d-lysine, be sure to rinse plates before plating cells as it is toxic to the cells when free in solution. Use the following procedure for coating plates:
  1. Pipet 50 ul/well of 50 mg/ml poly-d-lysine (in sterile water).
  2. Incubate plate at room temperature for 1 hour.
  3. Aspirate contents and pipet in 100 ul sterile water to wash wells.
  4. Aspirate water out and immediately plate cells. (Do not allow to dry as crystals may form causing the electrode on the bottom of the wells to lift.)

References

1. Atienza JM et al. (2006) Dynamic and label-free cell-based assays using the real-time cell electronic sensing system. Assay and Drug Development Technologies 4(5): 597-607.
2. Cooper, MA (2006) Non-optical screening platforms: the wave in label-free screening? Drug Discovery Today 11 (23-24): 1068-1074.
3. McGuinness, R (2007) Impedance-based cellular assay technologies: recent advances, future promise. Current Opinion in Pharmacology 7: 535-540.
4. Peters MF et al. (2007) Evaluation of Cellular Dielectric Spectroscopy, a whole-cell, label-free technology for drug discovery on Gi-coupled GPCRs. Journal of Biomolecular Screening 12(3): 312-319.
5. Yu et al. (2006) Real-time monitoring of morphological changes in living cells by electronic cell sensor arrays: an approach to study G protein-coupled receptors. Analytical Chemistry 78 (1): 35-43.
6. Leung, G et al. (2005) Cellular Dielectric Spectroscopy: A Label-Free Technology for Drug Discovery. JALA 10: 258-269.
7. CellKey™ System Assay Development Guide, MDS Analytical Technologies. 1021420 Rev B.

Contributing author to this chapter:

• John Proctor, Ph.D. - Application Scientist MDS Analytical Technologies