Computational protocol: Novel Fusion Protein Targeting Mitochondrial DNA Improves Pancreatic Islet Functional Potency and Islet Transplantation Outcomes

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Protocol publication

[…] Islets were dispersed into single-cell suspensions via incubation, with 0.05% Trypsin-ethylenediaminetetraacetic acid Mediatech, Inc.) for 5 min at 37 °C and passaged through a narrow-gauge pipette tip, resuspended in modified Krebs buffer (KRB) containing 3.3 mM glucose and 0.25% bovine serum albumin (BSA), and stained separately with probes for apoptosis (Annexin V PE, Invitrogen, Carlsbad, CA, USA; VAD-FMK FITC, Promega, Madison, WI, USA), and mitochondrial membrane polarity (MMP; JC-1, Invitrogen). Washed cells were analyzed on a 5-color FACS Calibur flow cytometer (Becton Dickenson, Franklin Lakes, NJ, USA). ToPro3 (Invitrogen) was added to samples immediately prior to analysis to identify necrotic cells. Data were analyzed using FlowJo software (Version 7.6.5) (Treestar, Inc., Ashland, OR, USA). [...] GraphPad Prism version 5.0 (GraphPad, San Diego, CA, USA) and SPSS (IBM SPSS Statistics for Windows, Version 22.0. Armonk, NY, USA) were used for statistical analysis. Differences between various groups were compared by use of 1-way analysis of variance (ANOVA) with the Bonferroni multiple-comparisons test for post hoc analyses. For data sets with nonnormal distribution, Kruskal-Wallis test and Dunn’s posttest correction were applied. All data are shown as mean ± standard error of the mean. Time to cure after transplant analyses were performed with Kaplan–Meier survival analysis using the log-rank test followed by pairwise comparisons to determine statistical difference between the groups. The level of statistical significance was defined as a P < 0.05. […]

Pipeline specifications

Software tools FlowJo, SPSS
Applications Miscellaneous, Flow cytometry