METHODS

Isolated pancreatic acini were prepared by a modification of the method of Amsterdam and Jamieson (1, 2) as used previously by us to prepare dissociated pancreatic a&nar cells (28). Starting-material was 0.9-1, 2 g of pancreas obtained from 7 to 10 male white Swiss mice (20-24 g) or from 1 to 2 male Sprague-Dawley rats (250-350 g). Animals were fasted overnight (14-18 h) prior to use, decapitated between 9: OO and 1 1: oo AM, and exsangu inated prior to removal of the pancreas. The basic medium used for isolation was a modified Krebs-Henseleit bicarbonate medium (KHB) to which soybean trypsin inhibitor and minimal Eagles medium amino acid supplement were added (&). Dissociation medium also contained 60-75 U/ml of purified collagenase, 0.03-0.05 mg/ml chymotrypsin (both from Worthington Biochemicals), and 1.8 mg/ml hyaluronidase (Sigma type l), and has a Ca2+ content of 0.1 mM? l The basic modification used to produG isolated acini, the elimi-nation of the divalent cation chelation step, was suggested by Dr. J. D. Jamieson in January, 1976 at a GAP Conference of the Cystic Fibrosis Foundation. We have used the resulting preparation for a variety of studies of the control of pancreatic amylase release (8, 32) without a previous complete description of our acinar preparation. Due to our new findings on amylase release and those of Dr. Jamieson’s laboratory, mainly carried out on guinea pig pancreas, it seemed worthwhile to report a more complete description of our preparation at this time.

2 The source and purity of collagenase used is critical. Work reported here was carried out with collagenase from Worthington Biochemical(Freehold, NJ)(lots 57 H 464, 57 J 494, and 57 N 392) reported as containing clostripain as a contaminant at less than 6 U/mg. Two other batches(lot 57 K 558 and 57 M 359 P) that had clostripain levels of 24 and 7 U/mg, respectively, would dissociate acini but showed considerable formation of cellular blebs, a high basal rate of amylase release, and a poor response to secretagogues. That the poor response is due to clostripain contamination has been shown by Gunther et al.,(13) who further purified collagenase and then added back individual contaminants. 0363-6100/78/0000-0000 $01.25 Copyright 0 1978 the American Physiological Society I% 17