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/.

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.