Necrosis in pancreatic acini and AR42J cells was determined by the release of lactate dehydrogenase (LDH) into the incubation medium, as previously described (Gukovskaya et al., 1997 (link), 2002 (link); Gukovskaya and Pandol, 2004 (link); Sung et al., 2009 (link)), LDH activity was measured using Cytotoxicity Detection Kit (Roche Diagnostics, Indianapolis, IN, USA) according to the manufacturer’s protocol.
Quantification of necrosis in in vivo pancreatitis was performed on pancreatic tissue (collected after four hourly cerulein injections) sections stained with H&E. Images from multiple random, non-overlapping sections were captured under a high-power field (×400-magnification, 6–10 random fields per section) with a Nikon Eclipse E600 microscope equipped with a digital camera using the SPOT imaging software (Diagnostic Instruments, Sterling Heights, MI, USA). Cells with swollen cytoplasm, loss of plasma membrane integrity, and leakage of organelles into interstitium were considered necrotic. A total of at least 2000 acinar cells were counted on tissue sections from each animal and three to five animals per condition were counted.
Quantification of necrosis in in vivo pancreatitis was performed on pancreatic tissue (collected after four hourly cerulein injections) sections stained with H&E. Images from multiple random, non-overlapping sections were captured under a high-power field (×400-magnification, 6–10 random fields per section) with a Nikon Eclipse E600 microscope equipped with a digital camera using the SPOT imaging software (Diagnostic Instruments, Sterling Heights, MI, USA). Cells with swollen cytoplasm, loss of plasma membrane integrity, and leakage of organelles into interstitium were considered necrotic. A total of at least 2000 acinar cells were counted on tissue sections from each animal and three to five animals per condition were counted.
