Detecting Caspase-8 Activity in Retinas

Protein preparation for the detection of caspase 8 activity was performed as previously described.¹⁶ (link) Two retinas for each mouse were pooled and homogenized in lysis buffer (20 mM MOPS, pH 7.0, 2 mM EGTA, 5 mM EDTA, 0.1% Triton X-100) containing protease inhibitor (complete protease inhibitor tablet [11697498001; Roche, Indianapolis, IN, USA]) and then centrifuged at 10,000 g for 15 minutes at 4°C. Then, 150 µg protein was used for each load and Caspase 8 activity was assayed in duplicate using luminescent Caspase-Glo 8 Assay kit (G8201; Promega, Madison, WI, USA), and luminescence was detected with a plate reader luminometer (Turner Biosystems, Sunnyvale, CA, USA; n = 8 for P23H and P23H/Fas-lpr; and n = 6 for P23H, rd10, rd10/Fas-lpr, and C57).