Ldh cytotoxicity detection kit

For detection of islet/β-cell death in vitro, cells were exposed to hypoxia (1% O₂) for 24 or 48 h. Cells were harvested and washed with PBS containing 5 mM EDTA. After fixation in 70% ethanol at 4 °C for 2 h, cells were collected, re-suspended in PBS containing 250 μg/ml RNase A, and incubated at 37 °C for 30 min. After propidium iodide staining, 2 × 10⁵ cells per sample were analyzed by flow cytometry. The portion of the apoptotic cells was calculated according to the apoptotic peak in the cell distribution against the propidium iodide fluorescence per cell. Cell death was also measured using a Cell Death Detection ELISA Kit and the colorimetric assay in medium using a LDH cytotoxicity detection kit (Clontech) as suggested by manufacturer (Roche, Indianapolis, IN, USA). For detection of β-cell apoptosis inside the pancreas, pancreatic tissue sections were incubated with terminal deoxynucleotidyl transferase in the presence of Biotin-dUTP (Roche Diagnostics). TUNEL-positive cells were revealed with Alexa Fluor 488-Streptavidin. Sections were double stained for insulin to distinguish β cells. Images were obtained by confocal microscopy. The numbers and fraction of TUNEL⁺ cells within an islet were calculated.

IL-1β release was measured by ELISA (eBiosciences). Cell death was quantified by measuring LDH release using the LDH Cytotoxicity detection kit (TaKaRa Clontech). To normalize for spontaneous cell lysis, the percentage of cell death was calculated as follows: [(LDH sample)−(LDH negative control)]/[(LDH positive control)−(LDH negative control)] × 100.