Glomax multimode plate reader

To determine caspase activation upon application of Golgi stress, 10⁶ A549 or HCT116 cells were seeded in 10 cm dishes and left to adhere overnight. The following day, the medium was replaced with medium containing either vehicle or BFA. TRAIL was added 6 h later. Twenty four hours after the addition of BFA, the cells were collected by gentle scraping on ice, washed with PBS and lysed in 500 μl RIPA buffer supplemented with protease inhibitors. The cell suspension was then briefly sonicated, centrifuged at 14 000 r.p.m., and the cleared lysate split in two equal portions for IP. To IP caspase-8 or CLFAR, 20 μl of the appropriate antibody was conjugated to 100 μl washed protein G agarose beads (Invitrogen) for 2 h at 4 °C with constant rotation, followed by three washes with PBS to remove unbound antibody. The beads were then equally divided over the samples for overnight IP of either caspase-8 or CFLAR. The following day, the beads were washed again and 10 μl of the beads re-suspended in PBS was used for a caspase-GLO-8 assay (Promega), performed in duplicate. Luminescence signal was read on a Glomax Multi Mode plate reader (Promega), after 1 h.

Fluorimetric determination of intracellular ROS was performed using a DCFDA assay kit (Abcam). Harvested cells in a 96-well microplate were incubated with 25 µM DCFDA in 1X Buffer for 45 min in the dark at 37 °C. Upon removal of DCFDA solution, 100 μL/well of 1X Buffer or 1X PBS was added and the fluorescence was measured immediately. For toxicity assays, 100 μL/well of TBHP 150 µM solution was added and cells were incubated for 1 h in the dark at 37 °C. For each sample, 10,000 events were acquired and intracellular ROS formation, which results from oxidation of the reagent, was detected by fluorescence spectroscopy, with maximum excitation and emission spectra of 495 nm and 529 nm respectively. Cells were analyzed using a GloMax® multimode plate reader (Promega Corporation, Madison, WI). The data were analyzed in triplicate.