Caspase 8 assay kit

Caspase-3 activity in cell lysates after treatments was measured by means of a Caspase 3 Assay Kit (Sigma-Aldrich) according to the manufacturer’s protocol, by assessing Ac-DEVD-7-amido-4-methylcoumarin cleavage and subsequent increase in 7-amido-4-methylcoumarin (AMC) fluorescence in a fluorometric plate reader Ascent Fluoroskan (Thermo Fisher Scientific, Waltham, MA, USA; λ_ex = 360 nm, λ_em = 460 nm). The data are presented as averages ± standard deviation of AMC concentration increase rate normalized for mg of cellular protein, averages ± standard deviation.
Caspase-8 in cell lysates after treatments was measured by means of a colorimetric Caspase 8 Assay Kit (Sigma-Aldrich) according to the manufacturer’s protocol, by assessing Ac-IETD-p-Nitroaniline cleavage and increase in p-Nitroaniline (pNA) concentration in a spectrophotometric plate reader Multiskan Go 1510 (Thermo Fisher Scientific, Waltham, MA, USA) reading absorbance at λ_ex = 405 nm. The data are presented as averages ± standard deviation of pNA concentration increase rate normalized for mg of cellular protein, averages ± standard deviation.

Nar (purity ≥ 95%), Nir (purity ≥ 95%), and Qu (purity ≥ 95%), trypsin, in vitro toxicology assay kit, Caspase-3 assay kit and Caspase-8 assay kit were procured from Sigma-Aldrich (USA). RPMI and DMEM media, and FBS were purchased from Life Technologies Corporation (California, USA). Total RNA isolation kit was purchased from Qiagen (Hilden, Germany). CM Sepharose and Superdex 75 for Balsamin purification were procured from GE Healthcare (Buckinghamshire, UK). Reagents used for electrophoresis were purchased from Bio Rad Laboratories (California, USA). All other reagents were purchased from Sigma-Aldrich (USA) and were of analytical grade.