Washington Cell Function Analysis Core

Overview

The Cell Function Analysis Core facilitates the research of Diabetes Research Center affiliates by offering precise real time functional analysis of intact cells and primary tissue. Many measurements are made using a flow culture system that maintains cell viability while multiple parameters are assessed non-invasively using optical or radiological detection. With the goal of systematically studying the physiologic control of cell function in health and in diabetes, assays are offered that reflect bioenergetics, metabolism, cell signaling and biosynthetic/secretory function. The Cell Function Analysis Core also provides an islet isolation service, which gives affiliates easy access to primary tissue/cells ex vivo.

Services are offered at two locations:

Cell Function Analysis Core (UW South Lake Union Campus)

• Assays for the following parameters: lactate production, glucose production and uptake, oxygen consumption, cellular NADH, CO2 production, reductive state of cytochrome C, ROS production, mitochondrial membrane potential, ATP content, calcium and potassium metabolism. These assays have been optimized for measurements in islets, retina, stem cells, macrophages, lymphocytes, adipocytes, endothelial cells, neurons, hepatocytes, muscle and brain slices.
• Determination of of protein kinase A and C activities using state-of-the-art technology based on genetically encoded fluorescence activity biosensors. In addition, microfluidic technology that permits single cell analysis of metabolic and electrophysiologic parameters.
• Isolation and culture of pancreatic islets and other primary tissue for subsequent morphological and functional characterization. Services for specific for islet studies include measurement of insulin secretion and islet insulin content.

Cell Function Analysis Core (VA Puget Sound Health Care System)

• Isolation and culture of pancreatic islets and other primary tissue for subsequent morphological and functional characterization.
• Sophisticated metabolic phenotyping of mice allowing for quantification of glucose tolerance, insulin sensitivity and β cell function.

Services

Flow Culture System

This system is optimal for real time measurements of cellular function. It is currently optimized for pancreatic islets, retinal cells, stem cells, macrophages, adipocytes, endothelial cells, neurons/brain slices and hepatocytes

The following measures can be made at the single cell level:

• Calcium
• NAD(P)H
• Mitochondrial Membrane Potential
• Reactive Oxygen Species
• Insulin (Zn++)
• Oxygen
• Potassium
• Protein Kinase A activity
• Protein Kinase C activity

The following measurements can be made in bulk:

• Insulin secretion
• Oxygen consumption
• Cytochrome C
• Lactate production

Static Culture System

This system is preferable where a large number of conditions need to be analyzed in parallel, or measures need to be made in the absence of flow.

This system is also optimized for pancreatic islets, retinal cells, stem cells, macrophages, adipocytes, endothelial cells, neurons and hepatocytes.

The following measurements can be made:

• Insulin secretion and content
• Ion transport (e.g. K+ and Ca++)
• Glycolysis and CO2 production
• Lactate production

In addition, assessment of metabolite levels, binding studies and enzyme activity assays are also offered.

Pancreatic Islets

Investigators can obtain isolated islets from rats and mice. The Core has experience isolating islets from Fischer, Sprague-Dawley, Wistar and BB rats, and C57BL/6, DBA/2, db/db and other strains and genetically modified mice. Although it is strain-dependent, rodents yield about 700 islets per rat, and 200 islets per mouse. Once prepared, islets may be cultured under desired conditions and/or treatments until being either shipped or further analyzed by methods listed above.

The Core can also assist investigators with acquiring monkey islets from the Oregon National Primate Research Center and culturing the islets upon arrival.

Mouse In Vivo Metabolic Phenotyping

The Core offers in vivo metabolic phenotyping of mice to complement and extend the in vitro functional assessment of cells and tissues. These studies can be performed in conscious mice using a dual catheter system.

The following measurements can be made:.

• Hyperinsulinemic-euglycemic clamp
• Intraperitoneal glucose tolerance test
• Intravenous glucose tolerance test
• Oral glucose tolerance test
• Islet transplantation
• Thoracic duct lymph collection

Consultation and Training

Input on study design and data analysis, together with training in the techniques utilized by the Core are offered. Contact the Director, Ian Sweet or Associate Director, Sakeneh Zraika , for more details.