Las4000 imagequant imager

Unless otherwise noted, all reagents were reagent/molecular biology grade or better. Liberase TH was purchased from Roche. Gentamicin (#15710-072) was obtained from Life Technologies (Carlsbad, CA). Primary antibodies against pro-caspase 9 (#9508), pro-caspase 3 (#9665), cleaved caspase 3 (#9664), and pro/cleaved PARP1 (#9542) were purchased from Cell Signaling Technologies (Boston, MA). Horseradish peroxidase-linked secondary antibodies against mouse (#NXA931) and rabbit (#NA934V) were from GE Healthcare (Pittsburgh, PA). SDS-PAGE pre-cast gels, 0.2 μm PVDF membrane (#162-0177) and protein molecular weight standards (#161-0374) were purchased from Bio-Rad (Hercules, CA). Amersham ECL reagent and LAS4000 ImageQuant Imager were purchased from GE Healthcare (Pittsburgh, PA). For the membrane potential assay, MitoTracker Green was obtained from Life Technologies (Carlsbad, CA) and tetramethylrhodamine ethyl ester perchlorate (TMRE) was obtained from Sigma Aldrich (St. Louis, MO). Laser scanning confocal microscopy was performed using Lab-Tek II four-chamber #1.5 German coverglass (#155382) from Nunc/Thermo Fisher (Rochester, NY) and a Leica TCS SP2 AOBS system mounted on a Leica DM IRE2 inverted microscope (Mannheim, Germany).

Following MXT and/or PACAP treatments, U937 cell samples were centrifuged for 10 min at 125 RCF and the treatment media aspirated. U937 cellular proteins were extracted with 1x RIPA lysis buffer for 15 min at 4°C and centrifuged for 10 min at 10,000 RCF to yield cell lysate supernatants. The protein concentration in each sample was quantified using the BCA (bicinchoninic acid) protein assay. Equal amounts of protein were loaded onto gels (4–20% TGX, Bio-Rad, Hercules, CA), separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then wet transferred onto polyvinylidene difluoride membranes. Blots were stained with Ponceau S and imaged to confirm equal protein loading and uniform blotting across the sample lanes. Blots were blocked in Tris-buffered saline with 0.05% Tween 20 containing 3% bovine serum albumin (TBST) for 1 h. Western blots were probed with a primary antibody against cleaved caspase 3 (#9664; Cell Signaling, Boston, MA) overnight, washed three times with TBST, probed with a secondary antibody against rabbit IgG (#NA934V; GE Healthcare, Pittsburgh, PA) for 1 h, and washed three more times with TBST. Finally, the blots were developed using the Amersham ECL imaged/quantified using the reagent and LAS4000 ImageQuant imager (both from GE Healthcare).