Services*

Islet Preparation

Preparation of Rodent Islets:
The IFAC has experience with Fischer, Sprague-Dawley, Wistar and BB rat islets, and C57 Black mouse islets. Other strains have also been successfully used. Investigators provide animals for rodent islet isolations if specific strains are desired. Although it is strain-dependent, rodents yield about 700 islets/rat, and 200 islets/mouse.

Preparation of Monkey Islets:
Primate pancreata are obtained by the IFAC for islet isolation and Affiliate use. Macaca nemestrina (pigtail monkey) pancreata are available an average of 1-2 times monthly for islet isolations from the Northwest Primate Research Center. These islets have similarities with human islets not shared by rodent islets. The yield is quite variable but averages about 20,000 IE/preparation.

Human Islets:
Human islets are not prepared by the IFAC. Arrangements can be made for the IFAC to receive human islets in order to carry out studies for investigators. To do this, investigators requesting human islets should contact one or more of the following laboratories.

Southern California Islet Cell Resource Center
City of Hope National Medical Center
http://icr.coh.org/ Contact Autumn Tate

Islet Isolation Core
Children's Hospital of Pittsburgh
University of Pittsburgh
Division of Immunogenetics
Contact Dr. Rita Bottino

University of California
San Francisco, Diabetes Center
Contact Dr. Gregory Szot

Dispersed Islet Cells:
To facilitate experiments that require dispersed islet cells, islets are treated with trypsin and then mechanically disrupted. Cells can be attached to glass slides allowing for binding, secretion or imaging studies.

Islet and Cell Assessment

Flow Culture/Assessment of Islets:
A flow culture system has been developed that allows continuous and concomitant assessment of hormone secretion, metabolite release (lactate, pyruvate etc.), oxygen consumption, and cytochrome redox state of islets cells or tissue for up to 48 hours (1-3). In addition, it can temporally control the culture conditions of the islets including chemical and gaseous composition of media and temperature, and can accommodate non-recirculating, recirculating and co-culture modes. Studies have typically involved the study of the acute and chronic effects of fuels, pharmacologic agents, hormones, neurotransmitters, metabolic poisons, cytokines, as well as studies of hypoxia. A sample data set is shown for the effect of glucose, diazoxide and glibenclamide on islet metabolism.

Static Culture and Assessment of Islets:
As a complement to flow culture studies, culture and assessment of islets maintained statically are carried out in response to specific needs determined by the Affiliate. More conditions can be handled statically so this mode is ideal for dose responses, particularly when cells need to be harvested for acquisition of sample. Examples are insulin secretory studies (7), uptake and efflux of radioactively labeled compounds, particularly for evaluation of beta cell imaging agents (6), effects of oxygen tension on cell viability and metabolic rates, production of low levels of metabolites or signaling molecules in the media, and phenotyping functional characteristics of islets from genetically altered mice. Sample data sets are shown at illustrating the dose response of -ketoisocaproate on insulin secretion, and on the kinetics of binding of radiolabeled glibenclamide to INS-1 cells.

Single Islet or Cell Functional Imaging of Calcium and NAD(P)H:
Measurement of calcium is done after loading the islets with FURA1-AM (4 µM) for 30 min. Islets (5) are loaded into a Bioptechs temperature-controlled perifusion chamber (Bioptechs Inc, Butler, PA) and perifused using a Gilson peristaltic pump. The perifusion chamber is placed onto the stage of a Nikon TE200 Eclipse inverted fluorescence microscope; images of the emitted fluorescence at 510 nm are captured using a CoolSnap EZ digital camera (Roper Scientific GmbH, Germany) every minute during excitation at alternatively 340 nm and 380 nm (pulse duration = 40 ms) accomplished using a Lambda 10-B filter wheel (AutoMate Scientific, Inc. San Francisco, CA) and a Nikon Xenon lamp. Data acquisition is driven using a Dell computer loaded with Metafluor software (Molecular Devices, Sunnyvale CA). NADH is measured similarly to calcium, except there is no need for loading with dye and the excitation wavelength is 360 nm and emission wavelength is 460 nm. Sample data sets are shown for the effect of glucose and acetylcholine on calcium in islets, and the effect of glucose on NAD(P)H in HMECs.

* Protocols for all services may be requested by email.