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We demonstrate the use of liquid handling workstations for performing cell-based assays. The feasibility of scale-up and minimal manipulation required to perform these assays makes them candidates for implementation on a variety of automated workstations. This article specifically describes the adaptation of Promega cell-based assays to the Beckman Coulter Biomek® 2000 and FX workstations in both 96- and 384-well formats. |
Introduction
Cell-based assays have become a quick, lower-cost means to test hit or lead
compounds for toxic effects before proceeding with drug development. The term
“cell-based assay” describes any procedure that uses
live cells for the evaluation of effects on toxicity, viability, proliferation
or other specific cellular processes following the addition of a test compound.
Of particular interest in drug discovery is the ability to analyze ADME/Tox
(Absorption, Distribution, Metabolism, Elimination/Toxicity) properties for
compounds of interest. Cell-based assays provide a platform in which these
properties and processes can be tested.
The process of performing a cell-based assay generally includes plating
cells, equilibrating to culture conditions, adding a test compound, and
measuring output from treatment. These assays can be made easier by automating
the process. Depending on the needs and financial resources of the user, a
single step or multiple steps of the assay process can be adapted to an
automated workstation. These workstations, whether they are liquid handlers or
fully-integrated systems with incubators, shakers and plate readers, allow
increased throughput for sample processing. Automation also removes the
requirement for hands-on assay performance, resulting in less error and higher
reproducibility.
We have incorporated and validated several Promega cell-based assays on the
Beckman Coulter Biomek® 2000 and FX automated workstations in both
96- and 384-well formats to meet the varying needs of the automation user. Table
1 highlights the assays that we have automated and validated on the Biomek®
platforms. Using these cell-based assay reagents, we have automated the final
step
in the assay process, allowing the measurement of output following treatment.
The simple and scalable “add-mix-measure” cell viability and apoptosis assay
formats make these assays ideal for multiwell plates and high-throughput
screening on any workstation.
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Table 1. Promega Automated Cell-Based Assays. The assays listed are validated on the Biomek® 2000 and FX platforms in both 96- and 384-well formats. |
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Assay |
Type |
Assay |
Output |
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Apoptosis |
Caspase-3/7 |
Luminescent |
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Apoptosis |
Caspase-3/7 |
Fluorescent |
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Membrane |
LDH Release |
Fluorescent |
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Cell Viability |
ATP |
Luminescent |
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Cell Viability |
Metabolic Capacity/ |
Fluorescent | |
Automated Method Development
Automated, single-plate methods were developed for five Promega cell-based assays in 96- and 384-well format on the Biomek® 2000 and FX workstations. Each method is a computer program that the robot will follow in order to perform the assay. These instructions encompass all the liquid handling steps, physical manipulations of each assay plate across the deck and operation of any devices on the deck.
To perform a cell-based assay on an automated workstation, the deck of the
workstation is manually
set up by adding labware, reagents, tip boxes and the sample plate. Once the
method is started, the robot will perform all steps without the need for manual
intervention. Due to the simplicity of the assays being automated, and the
methods written for each assay, the number of deck positions required to perform
each assay is minimal. Typically, only 3 to 4 deck positions are required for
the single-plate Biomek® 2000 and FX methods (Figure
1). While this number may vary depending on the method, each automated assay
will fit onto any deck layout that a robotic platform may have. This will also
make it possible to create multiplate methods that are able to fit onto the
original deck layout without the need for stacking capabilities.
The automated cell-based assay methods we developed perform three steps on the robotic platform (Table 2). Additional steps, including assay plate incubations and recording data from fluorescent or luminescent assays, are performed offline (separate from the robotic platform). These steps can be automated, depending on the level of automation required on the robotic platform being used. This fact, along with the methods’ simplicity, and minimal deck requirements, allows these assays to meet the needs of low-, medium-, or high-throughput research laboratories.
Validated methods to run these assays and other chemistries are available for
download at www.promega.com/automethods/. In
addition, the hardware, labware requirements and instructions are available as
automated protocols and are available at www.promega.com/tbs/.
Table 2. General Method for Single-Plate Automated
Cell-Based Assays.
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Validation and Results
Each single-plate method was validated according to the performance criteria
for each assay. Validation of these methods provides confidence that the methods
have been thoroughly tested and will provide consistent, reproducible results in
any setting. Results indicate that these automated assays provide reproducible
data that often equals or exceeds that achieved when performing the assays
manually (data not shown). This also represents a time savings to researchers by
allowing them to proceed directly to sample processing.
The criteria that we use for validation include the sensitivity of each assay, as well as linearity across a set range of cell or purified enzyme concentrations. Assay robustness and reproducibility are tested as well using Z´-factor determination (1).
The Z´-factor is a statistical value used to determine an assay’s robustness and reproducibility by comparing its dynamic range to the data variation. A Z´-factor value of 1.0 indicates a perfect assay. A Z´-factor value greater than 0.5 indicates excellent assay quality.
The Z´-factor was determined for each automated assay. Analyses were completed in 96- and 384-well formats using the Biomek® 2000 and Biomek® FX platforms. The results from each analysis are shown in Table 3. All assays and formats had a Z´-factor greater than 0.5.
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Table 3. Z´-Factor for Automated Cell-Based Assays developed on the Biomek® 2000 and FX platforms. |
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Automated Assay |
Cell-Based Paltform |
Robotic Well Format |
Assay Plate Z´-Factor |
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Biomek® 2000 Biomek® FX |
96 |
0.63 |
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Biomek® 2000 Biomek® FX |
96 |
0.80 |
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Biomek® 2000 Biomek® FX |
96 |
0.56 |
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Biomek® 2000 Biomek® FX |
96 |
0.76 |
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Biomek® 2000 Biomek® FX |
96 |
0.88 |
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Summary
We have demonstrated the ability to automate Promega’s cell-based assays in
both 96- and 384-well formats. Z´-factors for each assay format were above 0.5,
indicating that they were all excellent assays.
The minimal deck requirements of the automated methods for these cell-based
assays make them flexible enough to meet the needs of low-, medium- or
high-throughput laboratories.
Reference
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Figure 1. General deck configurations for cell-based assays on the Beckman Coulter Biomek® 2000 and Biomek® FX. The deck configurations shown above are a general representation for cell-based assays on the Biomek® 2000 (Panel A) and FX (Panel B). Additional labware, such as tip boxes, reagent reservoirs, or assay plates, will be required depending on the assay and the individual method being performed. An orbital shaker is required with each deck layout. |
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