Notes

1. Identifying the appropriate source, amount, and type of collagenase to be used for islet isolation is the most challenging aspect of the isolation of islets from rodent pancreata. The activity of collagenase is highly variable and dependent on source, supplier, and specific lot. We routinely use Type XI collagenase (Sigma, St. Louis, MO), although many laboratories use type P collagenase from Boehringer-Mannheim (Indianapolis, IN). Both of these sources of collagenase are specifically designed for pancreas digestion. It is critical to assess the activity of individual lots of collagenase, as the activity is highly variable. It is best to test several different lots of collagenase before purchasing a large supply of a specific lot. The activity of each lot should be consistent throughout.

2. There are three important variables to consider when choosing a lot of collagenase: (1) the amount of collagenase required to fully digest the pancreas, (2) the length of time for the digestion, and (3) the amount of pancreas to be digested. It is best

Fig. 4. Ficoll gradient centrifugation. The yield of islet in each gradient interface can vary depending on the digestion. The top interface (11-20.5%) will provide the highest yield of mouse islets. The second interface (20.5-23%) will provide the highest yield of rat islets. It is important to note that for each animal type, islets will be present at both interfaces.

Fig. 4. Ficoll gradient centrifugation. The yield of islet in each gradient interface can vary depending on the digestion. The top interface (11-20.5%) will provide the highest yield of mouse islets. The second interface (20.5-23%) will provide the highest yield of rat islets. It is important to note that for each animal type, islets will be present at both interfaces.

to begin by testing several different combinations (amount of collagenase used for the digestion and time of digestion) and compare the resulting yield of islets with a known lot of enzyme. We routinely initiate our characterization of a new lot of collagenase by varying the amount of collagenase. We start with 12-16 mg collagenase per tube of minced pancreas. Using equivalent volumes of pancreatic tissue in each test tube will help ensure consistency between tubes. The time of digestion is also a very critical parameter in a successful islet isolation.

3. When testing a new lot of collagenase, we usually begin with a 3.5- to 4-min digestion period. If the time required to digest the pancreatic tissue exceeds 7-7.5 min, increase the amount of collagenase. This will decrease the digestion time and also decrease islet damage that may occur during the digestion. While testing various lots of collagenase, the tissue volume should remain close to 3 mL. This volume should also include the 1 mm of HBSS remaining on the top of the tissue. If the tissue volumes are a bit smaller or larger, adjust the digestion time accordingly.

4. The end point for the collagenase digestion is determined visually. A good end point will have no visible tissue chunks remaining. It should appear smooth with a "creamy" texture. When holding the tubes up to the light, translucent gelatinous "spotting" that will run down the sides of the tube should be apparent. If there are large tissue chunks, return to the tubes to the water bath and shake for another 30 s. Repeat until you obtain the desired visual end point. If the material appears smooth, with no chunks, but has no spotting, continue to shake the tubes by hand at room temperature until the spotting appears. This change can take anywhere from several seconds to a couple of minutes. An underdigested islet preparation is characterized by a high level of acinar or ductal tissue attached to the islets. An overdigested islet preparation will have small ducts and little to no acinar cells, and the islets will have rough edges (islets with rough edges may recover following an overnight culture). In extreme cases of overdigestion the islets will disintegrate into single cells following an overnight culture. A normal, healthy islet will appear round with smooth edges.

5. One source of potential problems with the Ficoll preparation method of islet isolation is poor gradients or aberrant migration of islets in the Ficoll step gradients. To reduce potential problems with the islet Ficoll gradients, it is recommended that the refractive index of the Ficoll dilutions be examined to determine if the stock solutions are at the proper density for islet isolation. The following table gives the correct index and density values for each dilution.

Ficoll_Index_Density

6. Using the above-outlined protocols, one can expect to obtain approx 300-600 islets from each rat pancreas. On average, the yield from a mouse pancreas is lower, on the order of 80-180 islets. The yield of islets from transgenic animals may vary depending on the transgene expressed in islets or the effects of the specific transgene or gene knockout on b-cell development.

7. Once islets have been isolated, a number of additional manipulations can be performed. Islets can be dispersed into individual cells. This is routinely performed by trypsin treatment, as outlined previously (5,6). It is also possible to purify individual endocrine cells from islets by fluorescence-activated cell sorting (FACS). The technology for FACS purification of b-cells and non-b-cells was originally developed by Pipeleer's laboratory (7; see also Chapter 2). We routinely obtain approx 1.2-2 X 106 purified b-cells and approx 5-8 X 105 non-b-cells from islets isolated from 10 rats. The purity of these preparations is dependent on the parameters of FACS sorting, but in most purifications, we obtain approx 90-95% pure b-cells. The non-b-cell preparations contain primarily a-cells (approx 60%) as well as some b-cells, endothelial cells, and fibroblasts. The use of FACS-purified b-cells provides a unique method to directly examine the function of primary b-cells in the absence of other islet cellular components (8).

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