β-cell techniques

Molecular Biology:

Reverse transcription-PCR (RT-PCR) and quantitative RT-PCR

Real-time PCR

Restriction enzyme digest of DNA fragments

RNA isolation

Cloning - vector manipulation (gene insertion, deletion and siRNA design)

PCR Amplification of cDNA, genomic DNA and plasmids

Single and double stranded DNA sequencing (using S-35 & P-32)

Oligonucleotide site-directed mutagenesis

Luciferase assay

DNA Isolation (mouse and rat tail plus other sources)

Dot blot hybridization (using P-32)

HLA DR, DQ typing of patients

Northern, southern and western blotting

Adenovirus’ production (overexpression and siRNA)

Determination of the ATP/ADP ratio in clonal cell lines and isolated rat islets

Immunocytochemistry and immunohistochemistry (e.g. assays for β-cell proliferation, apoptosis and

        β-cell mass)

Radioimmunoassays (RIA)

Transmission electron microscopy (TEM)

Cryogenic tissue slicing

Drug encapsulation into microspheres for slow release

high performance liquid chromatography (HPLC)

Mass spectroscopy and protein sequencing

Thin layer chromatography - Fractionation of lipids

Enzymatic assays (e.g. citrate lyase, citrate synthase, malate dehydrogenase, lactate dehydrogenase)

Metabolite measurements (enzymatically and using mass spectrometry)

 

In vivo:

Genotyping and colony maintenance of knockout mice (UCP2 (-/-) mice) and transgenic mice (muscle-specific        mutant human IGF1 receptor, CD36 overexpression mice)

Oral glucose tolerance test (OGTT) (with plasma sampling for insulin in mice and rats)

Intraperitoneal glucose tolerance test (ipGTT) (with plasma sampling for insulin in mice and rats)

Insulin tolerance test (ITT) (mice and rats)

Constructed and calibrated a perfused mouse/rat pancreas

Collaboration on combined perfused mouse pancreas and measuring electrical activity from distinct

        sites in the pancreas.

Rat and mouse islet isolation and dispersion

Rat and mouse islet perifusion

Animal handling (primarily in rats and mice; also pigs, dogs, sheep, rabbits, frogs) (e.g. sc and ip injections and oral gavage)

Various animal surgeries in rats, mice and dogs (e.g. cannulation of the carotid artery, portal vein & jugular artery in rats)

Femoral vein injections and blood sampling

Blood sampling from femoral vein, saphanous vein, orbital sinus, cardiac puncture.

 

Live cell assays:

Laser scanning confocal microscopy (live and fixed cells/ tissues).

Oxygen-sensitive microelectrode system for the detection of single-cell oxygen consumption.  This system can also detect ionic flux, reactive oxygen species, glucose utilization, nitric oxide at the single cell level in real time.  See a website on the technique!!!

Single cell and whole islet imaging.  Includes measurements of cytosolic Ca²⁺ (fura-2, fluo-4) and mitochondrial membrane potential determination (Rh123, JC-1, TMRE) plus other dyes.

 

Electrophysiology:

Patch-clamping (cell-attached, whole cell, nystatin perforated patch clamping)

Computer based video analysis system for cell movement; computer image analysis software and plotting software.

 

Human experimentation:

Preparing subjects for centrifugation, data acquisition, data analysis, eye movement analysis using real-time EL-MAR eyetracking and pupillometry system