Orca flash4 cmos digital camera

Cytoplasmic histone–complexed DNA fragments were determined in islets using the Cell Death ELISAplus (Roche), per the manufacturer’s protocol. Cell pellets were analyzed to detect apoptosis. ELISA absorbance was measured at 450 nm and normalized to total DNA content (SYBRGreen; Invitrogen) using an absorbance/fluorescence microplate reader (BioTek). TUNEL was performed on transduced human islets (Apoptag In situ Apoptosis Detection Kit; Millipore) as per the manufacturer’s protocol. Human islets were prepared for staining as previously described (11 (link)). TUNEL-stained samples were counter stained with insulin and GFP antibodies. Images were captured with an IX81 microscope (Olympus) using an ORCA Flash4 CMOS digital camera (Hamamatsu). Apoptotic transduced β cells (TUNEL⁺, insulin⁺, GFP⁺) were counted as a fraction of total transduced β cells (insulin⁺, GFP⁺). DAPI served as a nuclear DNA control.

Mitochondrial morphology and localization studies were performed on immunostained human islets or paraffin-embedded mouse pancreas tissue sections, prepared as previously described (11 (link), 13 (link)). Z-stack images were captured with an IX81 microscope (Olympus) using an ORCA Flash4 CMOS digital camera (Hamamatsu) and subjected to deconvolution (CellSens; Olympus). Colocalization analyses were performed using the Coloc2 plugin on ImageJ (NIH). Mitochondrial morphology was visualized using 3D-renderings generated with Imaris imaging software (Bitplane). Quantitative 3D assessments of mitochondrial morphology and network were performed using the MitoMap and MitoAnalyzer plugins on ImageJ (67 (link), 68 (link)).