2.5. Caspase-3/7—Activity Assay

Cells were seeded in 6-well plates at a density of 100,000 cells/well. After one day of recovery inhibitors were added 1 h prior to PM_2.5 or medium (control cells) and incubated for 24 h. Subsequently, doxorubicin (final concentration 1 µM) was added for another 24 h before cells were washed with phosphate buffered saline and suspended in lysis buffer (10 mM HEPES-KOH pH 7.9, 0.5 mM ethylene glycol-bis(ß-aminoethy ether)-N,N,N’,N’-tetraacetic acid (EGTA), 0.5 mM ethylenediaminetetraacetic acid (EDTA), 350 µM NaCl, 1 mM MgCl₂, 1% Nonidet P-40, 20% glycerol, 5 mM dithiothreitol, 2.5 mM phenylmethylsulfonyl fluoride, 14 μg/mL aprotinin). Cell lysis was caused by three freeze–thaw cycles by freezing in liquid nitrogen and thawing in a 40 °C water bath and subsequent vortexing. After centrifugation (13,000× g, 4 °C, 10 min) protein concentration was quantified using the Bradford reagent (Quick Start^TM Bradford 1 × dye reagent, Bio-Rad, Munich, Germany). The fluorescence emitted by the release of 7-amino-4-methylcoumarin (AMC) from the caspase-3/-7 substrate (Ac-DEVD-AMC, Alexis, Lörrach, Germany) was monitored in a Fluostar Optima plate reader at an excitation wavelength of 370 nm and an emission wavelength of 450 nm. Relative fluorescence unit (RFU) values were calculated via the ratio rate of the fluorescence increase and protein concentration. RFU sample values were referred to control cells and are given as fold increase values.