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- Wash the stained cells with PBS twice.
- Add 0.3 mL PBS, protect samples from light, and measure the
FITC fl uorescence (FL1-H) with a fl ow cytometer, evaluating
the percentage of cells positive for CK18 cleavage in control
and apoptotic samples ( see Note 38 ). - Induce apoptosis in cells by desired method. For statistical ana-
lyzes, perform the experiment in triplicate wells. Concurrently
incubate triplicate control cultures without induction. - Harvest 2.5–5 × 10^5 cells from each well and centrifuge for
5 min at 150 × g , 4 °C. - Wash cells in PBS and centrifuge for an additional 5 min at
150 × g , 4 °C. - Discard supernatant. Resuspend each pellet in 30 μL cold PBS
and transfer into individual wells of an opaque 96-well assay
plate on ice ( see Note 39 ). - Pipette 5 μL of each sample for determination of protein con-
centration using the BCA ( see step 5 in Subheading 3.1.1 ). - Snap-freeze the samples in the plate by having it fl oating on
liquid nitrogen or incubate the plate at −20 °C for 30 min
( see Note 40 ). - Set up the computer control for microplate reader. Set tem-
perature to 37 °C. - Prepare complete 1× caspase activity sample buffer (2.5 mL
caspase activity stock buffer, 12.5 μL 1 M DTT, 2.5 μL
0.1 % NP40). This volume will be suffi cient for analyzing 50
samples. Immediately before use, add 2.5 μL of the caspase
substrate of interest. - Transfer the sample plate to room temperature and dispense
50 μL of complete sample buffer to each test well. Place the
plate in the microplate reader. - Read samples. The maximum absorption for AMC is 354 nm;
fl uorometric detection for AMC cleaved from peptide is at
excitation 380 nm and emission 460 nm. - The general working concentration of caspase inhibitors is
10–20 μM. Incubate cells for 1–2 h before inducing apoptosis
by the method of choice. It is recommended to concurrently
incubate control cultures without induction agent and inhibi-
tor, containing only induction agent and containing only
inhibitor ( see Note 41 ). - Proceed to analysis ( see Note 42 ).
3.5 Caspase Activity
Measurement
in Peptide
Cleavage Assay
3.5.1 Sample
Preparation
3.5.2 Caspase Activity
Measurement
3.6 Inhibition
of Caspase Activity
in Cell Cultures
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