Automated Cell-Based Assay to Determine Intracellular Caspase 3/7 Activities Using the CellProbe™ Caspase-3/7 Whole Cell Assay Kit

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SAMI® Workstation EX Software was used to develop a Biomek® Assay Workstation protocol for processing up to ten 96-well cell-based assay plates. The system consists of Biomek NX Multichannel unit, Cytomat 6000, Cytomat 2C, and integrated DTX 880 Multimode Detector (Figures 1 A, B and C, respectively). Details of the method and system are discussed below. Caspase 3/7 activity in HepG2 and HeLa cell lines was induced using staurosporine at different concentrations for four hours.

Hardware Setup

Figure 1A. Biomek NX Multichannel

Figure 1 B. Cytomat 6000 and DTX 880 Multimode Detector

Figure 1 C. Cytomat 2C

The Cytomat was configured to have tip boxes in Cytomat 6000 (Cytomat 1) and cell plates in Cytomat 2C (Cytomat 2) as shown in Figures 2 A and B.

Figure 3. Deck

The deck was configured to have inducer reagent and CellProbe substrate reagent on the deck prior to starting the method. Tip boxes and cell plates are brought to the deck according to SAMI EX Software scheduling as shown in Figure 3.

SAMI EX Software Method

A method was generated using the SAMI Workstation EX software to process up to 10 96-well cell plates. All transfer steps for adding inducer and substrate reagents plate were done on the deck scheduled by the SAMI EX Software (Figure 4). No user intervention was required once the method has started. The entire method was 5.5 hours.

Click to enlarge image

Figure 4. 1) Apoptosis induction, add inducer reagent to cell plates. (2) Incubation for four hours in Cytomat 2C at 37º C 5% CO2. (3) Add CellProbe substrate to cell plates. (4) Light sensitive CellProbe substrate is protected by dark lid with lid opened not exceeding 2 min 20s. This is controlled by "max time" function. (5) Incubation for one hour in Cytomat 2C at 37º C 5% CO2. (6) Read on DTX 880 at excitation 499 nm and emission 525 nm.

Data: Reproducibility Experiment

The results from this analysis indicate excellent and stable signal measured across all 10 plates, including Z' Factor and Signal to Baseline (Figure 5). The Z' factor is a statistical parameter use in evaluation and validation of high throughput screening assays.

Figure 5. The HTS assay design is excellent when the Z' factor value is between 1 and 0.5. In this case, there is a large separation of data points between the baseline and the positive signal. When the Z' factor value is between 0.5 and 0, there is only a small separation between the base line and the positive signal. The HTS assay design needs to be improved. In a "yes/no" assay the Z' factor is 0, indicating that there is insignificant separation of baseline and positive signal.

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For comments or questions about T³ Update, please contact David Daniels, Ph.D., editor.

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