Monitor toxic effects of compounds at high throughput capacities, or one sample at a time, in as little as 15 minutes to 1 hour, with Caspase-Glo^® Assays.

The Caspase-Glo Assays are homogeneous luminescent Assays that measure caspase-3/7, caspase-8 or caspase-9 activities.

Homogeneous "add-mix-measure" protocol means easy automation. The assays have been automated on Beckman Coulter's Biomek® 2000 and FX automated workstations in both 96- and 384-well formats with cells or purified enzyme. Both methods are available here. Achieve maximum sensitivity in as little as 15 minutes to 1 hour. No sample preparation or manipulation required, and no extended incubation times are necessary.

Figure 1. The Caspase-Glo Assay Protocol

Save on Cell Culture or Caspase enzyme use. The luminescent Caspase-Glo 3/7 Assay is more sensitive than fluorescent-based caspase assays, with a detection limit of 0.1pg recombinant caspase-3 enzyme. Sensitivity of less than 50 apotopic cells per well.

Figure 2. The Caspase-Glo 3/7 Assay provides superior sensitivity and linearity over a broad range of cell numbers.

Sensitivity several orders of magnitude greater than fluorescence-based assays for Caspase-Glo 8 and Caspase-Glo 9 Assays.

Figure 3. Caspase-Glo 8 and -9 are more sensitive than comparable fluorescent assays. In these examples, recombinant caspase-8 or -9 were two-fold serially diluted in 10mM Hepes, pH 7.4, with 0.1% Prionex. Caspase-Glo 8 or -9 reagent or respective AFC-labeled fluorescent substrated diluted in Caspase-Glo Buffer with exogeneously added DTT or 10mM, were added to the cells in equal volumes. Luminescence or fluorescence signal was measured at 1 hour on a BMG Fluostar or Cytofluor II, respectively. Square symbols represent Caspase-Glo signal-to-noise ratios whereas triangle symbols represent signal-to-noise ratios derived from their fluorescent counterparts. The dotted line denotes a S:N ratio of 3, indicating a limit of detection equal to 3 standard deviations above background. For these caspase titrations, the Caspase-Glo Assays have not reached their limits of detection. The lowest doses are well below the limit of detection for the fluorescent assays.

High predictive value. Caspase-Glo Assays can produce excellent Z′ values and are suitable for HTS applications. Use fewer cells when monitoring apoptosis, and less recombinant caspase when screening for caspase inhibitors.

Figure 4. The Caspase-Glo Assays can produce excellent Z′-factor values. Z′-factor = 0.92 for Caspase-Glo^® 3/7. A Z′-factor was calculated using recombinant caspase-3 and a non-caspase control in two 96-well plates.

Figure 5. The Caspase-Glo Assays are suitable for HTS applications. Z′-factors were calculated using recombinant  caspase-8 or -9 (Biomol) at 10U/ml and with no-caspase buffer blanks. Assays were performed in a total volume of 200µl in a single, solid white 96-well plate (48 wells/sample and control). Half of the wells of the plate contained buffer and purified caspase-8 or -9 (+ caspase-8 or -9), and half of the wells contained buffer only with no purified caspase (-caspase-8 or -9). Z′-factors were 0.957 and 0.903, respectively for the assays. One unit of caspase is the amount of enzyme require to cleave 1pmol of substrate (Ac-LETD-pNA or Ac-LEHD-pNA) per minute at 30°C, per manufacturer's unit definition.

View Online Catalog Information
Caspase-Glo^®3/7 Assay
Caspase-Glo^® 8 Assay
Caspase-Glo^® 9 Assay