Caspase assay kit

Caspases were induced in RAW264.7 by treating cells with 2 μg/ml of PE for 4 hours. Caspases were extracted and their activity was measured using Caspase Assay Kit (Sigma-Aldrich) with or without drugs.
The FRET reaction was performed in 96 well plates, and each reaction contained 16 μM substrate peptides (Peptides International) conjugated with a 7-amino-4-methylcoumarin group at the N-terminus and acetyl group at the C-terminus. The amino acid sequences of substrates were: DNLD for caspase-3, DQTD for caspases-3 and -7, and VEID for caspase-6. CCL compounds were tested at the final concentration of 33 μM, as pilot testing indicated that CCL screen at 16 μM would not yield a sufficient number of multiplex hits. Reactions were initiated by adding caspase – containing lysate to a final concentration of 6 μg/ml. Kinetic measurements were obtained at 37 °C every 5 minutes for 2 hours using a fluorescent plate reader. Excitation and emission wavelengths were 360 nm and 460 nm, respectively, with a cutoff wavelength of 365 nm. Rates of reactions were quantified by the Microsoft Excel LINEST function. A BioVision kit was used to test Bithionol’s ability to inhibit FRET reactions of purified recombinant human caspases-1 through 10. One unit of caspase was used in a single FRET reaction.

ATO was purchased from Fischer Scientific (Pittsburgh, PA). Mitochondrial isolation kit, Caspase assay kit, protease inhibitor and Glutathione assay kit were obtained from Sigma-Aldrich (St. Louis, MO). Anti-Cytochrome C, anti-Bax and anti-Bcl2 were purchased by Cell Signaling Technology (Danvers, MA). Lipid peroxidation kit and caspase 3 kit were obtained from Abcam (Cambridge, MA). Mitotracker red, Hoechst 33342, Alexa fluor 568 and Alexa fluor 568 were purchased from Life Technologies (Grand Island/NY).