Description
Caspase 3/7 Assay Kit (Fluorometric) (BA0020) (BA0020)
The Caspase 3/7 Assay Kit (Fluorometric) (SKU: BA0020) provides a convenient fluorimetric method for measuring caspase-3 activity as a marker of apoptosis in biological samples. Caspases are aspartate-specific cysteinyl proteases that play a central role in apoptosis, and caspase-3 is a key biomarker for assessing apoptosis and understanding its induction. In the assay, the specific substrate N-Ac-DEVD-AFC is cleaved by active caspase-3 to form a highly fluorescent product, with fluorescence intensity (excitation 400 nm, emission 490 nm) proportional to caspase-3 activity. The homogeneous mix-incubate-measure format works directly with cells cultured in black clear-bottom 96-well plates as well as with prepared cell or tissue lysates. It is non-radioactive, readily automated, and ideal for high-throughput screening of apoptosis inducers and inhibitors.
| Product Name: | Caspase 3/7 Assay Kit (Fluorometric) (BA0020) |
| SKU: | BA0020 |
| Detection Method: | Fluorometric (Ex 400 nm / Em 490 nm) |
| Sample Type: | ['Cultured cells', 'Cell lysate', 'Tissue lysate'] |
| Species Reactivity: | All |
| Assay Time: | 60 minutes incubation |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | -20°C |
| Shelf Life: | 12 months after receipt |
| Shipping: | Room Temperature |
Caspases are members of the aspartate-specific cysteinyl protease family that play a central role in apoptosis. Apoptosis is involved in a variety of physiological and pathological events, ranging from normal fetal development to diseases such as cancer, organ failure and neurodegenerative diseases. Caspase-3 is a key biomarker in the assessment of apoptosis and in understanding the mechanism of apoptosis induction. This kit provides a convenient means to measure caspase-3 activity in biological samples: a specific substrate (N-Ac-DEVD-AFC) is cleaved by active caspase-3, forming a highly fluorescent product, and the fluorescence intensity (excitation 400 nm, emission 490 nm) is proportional to the caspase-3 activity.
- Safe. Non-radioactive assay.
- Convenient and high-throughput. Homogeneous mix-incubate-measure type assay that can be readily automated on HTS liquid handling systems for processing thousands of samples per day.
- Determination of caspase-3 activity in cell and tissue lysate.
- HTS screening for apoptosis inducers and inhibitors.
Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Procedure |
| 1 | Seed 100 µL of 1,000 to 100,000 cells (depending on the cell line) into wells of a sterile black clear-bottom 96-well plate and incubate overnight at 37°C in a cell culture incubator. Duplicate assays are recommended for controls and samples. |
| 2 | Add 10 µL test compounds (e.g. apoptosis inducers or inhibitors) at the desired concentration, and 10 µL vehicle (the medium in which the test compound is dissolved) as a control, then incubate the cells for the desired period of time. |
| 3 | Bring all reagents to room temperature and briefly centrifuge the tubes. Prepare enough Working Reagent by mixing, for each well, 100 µL Assay Buffer, 2 µL Substrate and 2 µL DTT. |
| 4 | Remove the culture media from the assay wells: for adherent cells simply aspirate, and for suspension cells centrifuge for 5 min at 500 x g and carefully aspirate. Immediately add 100 µL Working Reagent to each assay well and mix completely by shaking the plate for 60 sec at 100-200 rpm; the Working Reagent lyses the cells and supports optimal caspase-3 activity. |
| 5 | Incubate the plate at 37°C for 60 min in the dark. |
| 6 | Read fluorescence intensity at excitation 400 nm and emission 490 nm. |
Subtract the fluorescence intensity values of the control wells from those of the sample wells. The resulting change in fluorescence (delta F) represents the relative caspase-3 activity. If the caspase-3 activity is low, increase the incubation time or use more cells.
| Component | Quantity | Storage |
| Assay Buffer | 12 mL | -20°C |
| Substrate | 240 µL | -20°C |
| DTT Solution | 240 µL | -20°C |
