Protein concentrations were determined using the

bicinchoninic acid protein assay kit. Samples were then mixed with loading buffer and run on a 15% sodium dodecyl sulfate–polyacrylamide gel. This gel was then transferred to a polyvinylidene fluoride membrane at 250 mA for 2 hours. The membrane was blocked in 5% milk for 1 hour and then incubated in primary (LC3 or activated caspase-3; Cell Signaling Technology) in 1% milk or phosphorylated selleck chemical p38 MAPK in 5% bovine serum albumin overnight. Membranes were in TBS-Tween 20 (TBST) for 30 minutes, then placed in secondary antibody linked to horseradish peroxidase for 1 hour and washed for 1 hour in TBST before being developed using a chemiluminescence substance (Thermo Scientific). For electron microscopy, mice were perfused with cold PBS, then with 2% paraformaldehyde

and 2% glutaraldehyde in 0.1 mol/L phosphate buffer (pH 7.4) and processed DAPT for transmission electron microscopy (TEM) as described.8 After dehydration, thin sections were stained with uranyl acetate and lead citrate for observation under a JEM 1011CX electron microscope (JEOL, Peabody, MA). Images were acquired digitally from a randomly selected pool of 10-15 fields under each condition. Fixed cells or tissue samples underwent terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining with the En Roche kit, per the manufacturer’s protocol. Images were taken with a Zeiss 510 inverted confocal microscope. Mitochondrial membrane potential was determined using the mitochondrial dye, JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocarbocyanine iodide; Molecular Probes, Eugene, OR). In the cytosol and mitochondria at low membrane

potential, the monomeric form of JC-1 fluoresces green (emission at 525 nm), whereas within the mitochondrial matrix at high membrane potentials, JC-1 forms aggregates that fluoresce red (emission at 590 nm). Samples were incubated with JC-1 at a final concentration of 1 μM at 37°C for the last 30 minutes of the experiment. Flow cytometry (Guava, Millipore) was used, and red and green fluorescence was determined. Results are expressed as the ratio of red:green fluorescence. Total cell counts were also obtained through the use Ribonucleotide reductase of flow cytometry. Cell Titer-Glo luminescent cell viability assay (Promega), per the manufacturer’s instructions, was used for the quantification of ATP content. Luminescence was measured using the SoftMaxPro ATPase Assay program on a Synergy Mx (Biotek) plate reader. C57BL/6 mice were randomized to sham operation or cecal ligation and puncture. Mice were sacrificed 8 or 20 hours after this insult, and liver tissue was collected. Induction of autophagy was determined using western blotting, immunohistochemistry, and TEM.