Assay Procedure:1. Induce apoptosis in cells by desired method. Concurrently incubate a control culture without induction. 2. Count cells and pellet 1-5x106 cells. 3. Resuspend cells in 50µl of chilled Cell Lysis Buffer and incubate cells on ice for 10 min. 4. Centrifuge for 1 min. in a microcentrifuge (10’000 x g). 5. Transfer supernatant to a fresh tube and assay protein concentration. 6. Dilute 50-200µg protein to 50µl Cell Lysis Buffer for each assay.7. Add 50µl of 2x Reaction Buffer containing 10mM DTT to each sample. 9. Add 5µl of the 4mM pNA conjugated substrates (200µM final conc.) into each tube individually and incubate at 37°C for 1-2 hours. 10. Read samples at 400 or 405nm in a microtiter plate reader, or spectrophotometer using a 100 µl micro quartz cuvet, or dilute sample to 1ml with Dilution Buffer and using regular cuvet (Note: Dilution of the samples proportionally decreases the reading). Fold-increase in caspase activity can be determined by comparing these results with the level of the uninduced control. Note: Background reading from cell lysates and buffers should be subtracted from the readings of both induced and the uninduced samples before calculating fold increase in caspase activity.
Assay Procedure:1. Induce apoptosis in cells by desired method. Concurrently incubate a control culture without induction. 2. Count cells and pellet 1-5x106 cells. 3. Resuspend cells in 50 µl of chilled Cell Lysis Buffer and incubate cells on ice for 10 min. Centrifuge for 1 min. in a microcentrifuge (10’000 x g). 5. Transfer supernatant to a fresh tube and assay protein concentration. 6. Dilute 100-300 µg protein to 50 µl Cell Lysis Buffer for each test. 7. Add 50 µl of 2x Reaction Buffer containing 10 mM DTT to each sample. 8. Add 5 µl of the 4 mM pNA conjugated substrates (200 µM final conc.) into each tube individually and incubate at 37°C for 1-2 hours. 9. Read samples at 400 or 405nm in a microtiter plate reader, or spectrophotometer using a 100 µl micro quartz cuvet (Sigma), or dilute sample to 1ml with Dilution Buffer and using regular cuvet (note: dilution of the samples proportionally decreases the reading). Fold-increase in caspase activity can be determined by comparing these results with the level of the uninduced control. Note: Background reading from cell lysates and buffers should be subtracted from the readings of both induced and the uninduced samples before calculating fold increase in caspase activity.
Assay Procedure:1. Induce apoptosis in cells by desired method. Concurrently incubate a control culture without induction.2. Count cells and pellet 1-5 x 106 cells or use 50-200 μg cell lysates if protein concentration has been measured. 3. Resuspend cells in 50 μl of chilled Cell Lysis Buffer. 4. Incubate cells on ice for 10 minutes. 5. Add 50 μl of 2x Reaction Buffer and 1 μl DTT to each sample.6. Add 5 μl of the 1 mM AFC conjugated substrates (50 μM final conc.) into each tube individually and incubate at 37°C for 1-2 hour. 7. Read samples in a fluorometer equipped with a 400 nm excitation filter and 505 nm emission filter. For a plate-reading set-up, transfer the samples to a 96-well plate. You may perform the entire assay directly in a 96-well plate. Fold-increase in caspase activity can be determined by comparing these results with the level of the uninduced control.
