The Caspase 3 Assay Kit, BioAssay™ provides a convenient means to measure caspase-3 activity in biological samples. In the assay, a specific substrate (N-Ac-DEVD-AFC) is cleaved by active caspase-3, forming a highly fluorescent product. The fluorescence intensity (lexc/em=400/490nm) is proportional to the caspace-3 activity.

Safe. Non-radioactive assay. Convenient and high-throughput. Homogeneous "mix-incubate-measure" type assay. Can be readily automated on HTS liquid handling systems for processing thousands of samples per day.

Determination of caspase-3 activity in cell and cell lysate. HTS screening for apoptosis inducers and inhibitors.

Assay Buffer: 12ml Substrate: 240ul DTT Solution: 240ul Storage conditions: This product is shipped at room temperature. Upon delivery, store all reagents at -20°C. Shelf life of 12 months after receipt. Precautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.

Note: The following procedure is for standard assays in a 96-well plate. If cells are cultured in plates other than a 96-well plate, it is necessary to prepare the cell lysate (See General Considerations). Duplicate assays are recommended for controls and samples. 1. Cell culture. Seed 100ul of 1,000 to 100,000 cells into wells of a sterile black clear-bottom 96-well plate (Note: The cell number to be used depends on the cell line). Incubate overnight at 37°C in a cell culture incubator. 2. Cell treatment. Add 10ul test compounds (e.g. apoptosis inducers or inhibitors) at desired concentration, and 10ul vehicle (medium in which the test compound is dissolved) as a control. Incubate cells for a desired period of time. 3. Caspase-3 Assay. Prior to assay, bring all reagents to room temperature. Briefly centrifuge tubes. Prepare enough Working Reagent for all assay wells. For each well, mix 100ul Assay Buffer, 2ul Substrate and 2ul DTT. Remove culture media from assay wells. For adherent cells, simply aspirate the culture media from wells. For suspension cells, centrifuge cells for 5 min at 500 x g, carefully remove media by aspiration. Immediately add 100ul Working Reagent to each assay well. Mix the reagents completely by shaking the plate for 60 sec at 100-200 rpm on a plate shaker. The Working Reagent lyses cells and supports optimal caspase-3 activity. Incubate plate at 37°C for 60 min in the dark. 4. Read fluorescence intensity at lexc/em=400/490 nm. Note: If the caspase-3 activity is low, increase the incubation time, or use more cells.

Subtract the fluorescence intensity values from that of the control wells. The DF values represent the relative caspase-3 activity.

For cells not cultured in a 96-well plate, cell lysates are prepared separately and used for the caspase assay. After cells have been treated with test compounds for the desired period of treatment, remove culture medium. For adherent cells simply aspirate the culture medium. For suspension cells centrifuge cells at 500 x g for 5 min and aspirate the medium. Lyse cells by adding 300ul per 106 cells of 50mM Hepes (pH 7.2), 100mM NaCl, 0.5 % (v/v) Triton X-100. Shake the cell suspension on for 30 min at 4°C. Centrifuge the cell suspension at 2,500 x g for 10 min at 4°C. Transfer supernatants to clean tubes. If not assayed on the same day, lysates can be stored at -80°C for one month. To assay, add 50ul of sample lysates into separate wells of a black 96-well plate. Immediately add 100ul Working Reagent to each assay well. Incubate plate at 37C for 60 min in the dark. Read fluorescence intensity lexc/em=400/490 nm.

Pipetting devices, centrifuge tubes, black flat bottom 96-well plates and plate reader.