Computational protocol: Sphingosine kinase 1-interacting protein is a novel regulator of glucose-stimulated insulin secretion

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Protocol publication

[…] Tissue and cells protein was extracted by homogenization in lysis buffer (Sigma). Twenty micrograms of total protein were resolved by SDS-PAGE on 4–12% acrylamide gels (Invitrogen) and transferred to PVDF membranes (Invitrogen), followed by immunoblotting with antibody. Quantification of bands on western blot was accomplished by scanning the blots, then determining the densities of the bands using ImageJ software (National Institutes of Health, Bethesda, Maryland, USA). [...] Isolated islets were cultured in RPMI 1640 with 11.1 mM glucose overnight, and observed under incubation using incubator two-photon excitation microscopy (Olympus LCV110-MPE) at 37 °C in a humidified atmosphere (5% CO2 and 95% air). Two-photon excitation was effected at 1040 nm, the mCherry fluorescence was measured at 575–650 nm, the GFP fluorescence was measured at 515–560 nm, and negative control fluorescence was measured at 460–495 nm. Digital images (512 × 512 pixels) were taken and analyzed using IMARIS software. […]

Pipeline specifications

Software tools ImageJ, Imaris
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Mus musculus
Chemicals Adenosine Triphosphate, Glucose, Sphingosine