163- Anti-Cytokeratin 18 M30 CytoDEATH Monoclonal Antibody,
FITC Conjugated ( Roche Applied Science ). - Antibody dilution buffer: PBS containing 1 % BSA.
- FACS instrument.
- PBS, pH 7.4.
- Pierce™ BCA Protein Assay Kit (Thermo Scientifi c) or similar.
- Caspase activity buffer I, stock (analysis of caspase-1, caspase-
3/-7, caspase-6, and caspase-8 activities): 100 mM HEPES
(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), pH 7.5,
10 % (w/v) sucrose, 0.1 % (w/v) CHAPS (3-[(3-Cholamidopropyl)
dimethylammonio]-1-propanesulfonate). - Caspase activity buffer II (analysis of caspase-2 and caspase-9
activities): 0.1 M MES (2-(N-Morpholino) ethanesulfonic
acid), pH 6.5, 10 % (w/v) PEG (Polyethylene glycol), 0.1 %
(w/v) CHAPS ( see Note 14 ). - 1 M DTT (dithiothreitol): Dissolve 309 mg DTT in a fi nal
volume of 2 mL 0.01 M sodium acetate, pH 5.2. Sterilize by
fi ltration, make aliquots, and store at −20 °C. - 0.1 % (v/v) NP-40 (Nonidet P-40): Add 1 μL of NP-40 to
1 mL water, vortex, and keep at 4 °C. - Caspase substrate stock solutions: Prepare stock solutions of
each specifi c caspase substrate to be tested in DMSO as
follows:
Caspase-1 (Ac-YVAD-AMC) 100 μM.
Caspase-2 (Ac-VDVAD-AMC) 100 μM.
Caspase-3/-7 (Ac-DEVD-AMC) 40 μM.
Caspase-6 (Ac-VEID-AMC) 100 μM.
Caspase-8 (Ac-IETD-AMC) 100 μM.
Caspase-9 (Ac-LEHD-AMC) 100 μM.
Make aliquots and store at −20 °C. - Fluoroscan Ascent Microplate Fluorometer (Thermo Scientifi c)
or similar. - Opaque 96-well assay plate (e.g., Corning Inc.)
- Gen5 (Bio Tec) or similar software for acquisition and data
processing.
2.4.2 Detection of the CK
18 Neoepitope
2.5 Caspase Activity
Measurement in
Peptide
Cleavage Assay
2.5.1 Sample
Preparation
2.5.2 Caspase Activity
Measurement
Caspases in Mammalian Cell Cultures