Ligand Characterization Using Microphysiometry

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

This unit describes the use of a Cytosensor microphysiometer for functional characterization of an agonist and antagonist to a G protein?coupled receptor, the muscarinic M1 receptor. Concentration?response profiles are used to calculate values for the EC50 of the response of cells to the agonist and the pA2 value for the antagonist. Support protocols describe optimization of two aspects of this procedure: the duration of ligand exposure at a given concentration and the length of recovery time between the administration of two different concentrations of ligand to minimize the impact of desensitization. The Cytosensor microphysiometer allows the measurement of receptor activation in both adherent cells, such as the M1WT3 cells used here or in suspension cultures.

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Basic Protocol 1: Characterizing the Response of Adherent M1‐Transfected CHO Cells Using the Cytosensor Microphysiometer
Alternate Protocol 1: Characterizing the Response of Nonadherent Cells Using the Cytosensor Microphysiometer
Support Protocol 1: Optimizing Ligand Exposure Time
Support Protocol 2: Optimizing Recovery Time
Reagents and Solutions
Commentary
Literature Cited
Figures

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Materials

Basic Protocol 1: Characterizing the Response of Adherent M1‐Transfected CHO Cells Using the Cytosensor Microphysiometer

Materials

Confluent cultures of adherent M₁ ‐transfected CHO cells (M1WT3, ATCC CRL‐1985) grown in 25‐cm² flasks
0.02% (w/v) EDTA
Growth medium (see recipe )
Running medium (see recipe ), freshly prepared
Carbachol (RBI)
Pirenzepine dihydrochloride (RBI)
Capsule cups (Molecular Devices)
Blunt forceps, sterile
Capsule spacers (Molecular Devices)
Capsule inserts (Molecular Devices)
Sensor chambers, sterile
Cytosensor microphysiometer workstation, sterile
Cytosensor system software
50‐ and 15‐ml polypropylene tubes

NOTE: All solutions and equipment coming into contact with live cells must be sterile, and proper aseptic technique should be used accordingly.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO₂ incubator unless otherwise specified.

Alternate Protocol 1: Characterizing the Response of Nonadherent Cells Using the Cytosensor Microphysiometer

Agarose entrapment medium (Molecular Devices)
Cultures of nonadherent cells expressing the receptor of interest (∼1 to 5 × 10⁶ cells total)
Heating block or hot plate at >70°C, or microwave oven
Incubator or heating block at 37°C

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Figures

Figure 7.8.1 Assembly of the microphysiometer cell capsule and sensor chamber.

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Figure 7.8.2 (A ) Screen shot from Cytosoft, the Cytosensor system software. Acidification‐rate data from eight chambers of M1WT3 cells in response to sequential incubations with gradually increasing concentrations of carbachol ± antagonist, using a similar protocol to that outlined in the text. Duplicate chambers are run in the presence of 0 nM, 30 nM, 300 nM and 3 µM pirenzepine, a selective M₁ receptor antagonist. (B ) Expanded screen shot of the acidification rate response to 10 µM carbachol (added during the 70‐sec period shown by the black vertical bar) in the absence and presence of pirenzepine (PZP). The acidification rate traces have been normalized in this figure to a baseline period selected just prior to carbachol addition.

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Figure 7.8.3 Calculated concentration‐response curves from the data shown in Figure . Each data point represents the mean of the two replicates from a single experiment.

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Figure 7.8.4 Schild plot calculated from the acidification‐rate data. Solid line represents the experimental data points. The broken line is the linear regression through these points. The intercept on the x ‐axis, the pA₂ value, is 8.0.

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Literature Cited

Literature Cited
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