Selected cytokines, the limits of detection, and the coefficients selleck kinase inhibitor of variability (intra-assay percent coefficient of variation [%CV] and interassay %CV) of the cytokine/chemokine are reported in Table S5 in the supplemental material. Evaluation of cell death following infection (LIVE/DEAD assay). The viability of islet cells after infection with human viruses was measured using the LIVE/DEAD cell assay kit (L-3224; Molecular Probes, Inc., Leiden, The Netherlands). The assay is based on the simultaneous determination of live and dead cells with two fluorescent probes. Live cells are stained green by calcein due to their esterase activity, and nuclei of dead cells are stained red by ethidium homodimer 1. Islets harvested after 5 days of culture were further enzymatically digested into single cells with trypsin-like enzyme (12605-01; TrypLE Express; Invitrogen, Carlsbad, CA).

According to the manufacturer’s instructions, single cells were incubated with the labeling solution for 30 min at room temperature in the dark, cytocentrifuged onto glass slides, and assessed with a Carl Zeiss Axiovert 135TV fluorescence microscope. Analysis of dead cells was performed on cytospin preparations using IN Cell Investigator software (GE Healthcare United Kingdom Ltd.). Positive cells in each category were quantified with 10 systematically random fields. Statistical analysis. Data were generally expressed as mean �� standard deviation or median (minimum-maximum). Differences between parameters were evaluated using Student’s t test when parameters were normally distributed and a Mann-Whitney U test when parameters were not normally distributed. Kaplan-Meier and/or Cox regression analysis was used to analyze the incidence of events during the time. A P value of less than 0.05 was considered an indicator of statistical significance. Analysis of data was done using the SPSS statistical package for Windows (SPSS Inc., Chicago, IL). Nucleotide sequence accession Drug_discovery numbers.

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