Collagenase containing solution

Pancreatic acinar cells were isolated as previously described [9 (link)]. Briefly, animals were sacrificed according to the Animal Scientific Procedures Act, 1986 and approved by the Ethical Review Committee of Cardiff University. After dissection, the pancreas was digested using collagenase-containing solution (200 U ml⁻¹, Worthington, UK) and incubated in a 37°C water bath for 14–15 min. The extracellular solution contained: 140 mM NaCl, 4.7 mM KCl, 10 mM Hepes, 1 mM MgCl₂, 10 mM glucose, pH 7.2, and CaCl₂ (0–2 mM as described in the text).

Cells were isolated as previously described (18 (link)). After dissection, the pancreas was digested using collagenase-containing solution (200 IU/ml, Worthington) and incubated in a 37°C water bath for 14 to 15 minutes. The extracellular solution contained the following: 140 mM NaCl, 4.7 mM KCl, 10 mM HEPES, 1 mM MgCl₂, 10 mM glucose, pH 7.3, and 1 mM CaCl₂. Osmolarity was checked by Osmomat 030. All in vitro experiments were conducted using this solution unless otherwise stated.

Pancreatic acinar cells (single, or clusters of two or three cells) were isolated from the pancreas of adult C57BL/6 mice (control wt and mutant male or female mice with C57Bl/6JJmsSIc origin) as described previously [4] (link). Briefly, animals were killed according to UK Schedule 1 regulations and after dissection the pancreas was transferred into a collagenase-containing solution (200 U/ml, Worthington, UK), and incubated at 37 °C water bath for 14–15 min. After digestion, the tissue was mechanically disrupted in Na-Hepes based extracellular buffer, containing (mM): NaCl, 140; KCl, 4.7; Hepes KOH, 10; MgCl₂, 1; glucose 10; CaCl₂ 1; pH 7.2. Cells were then loaded with an appropriate fluorescent dye (Fluo-5N AM or Fluo-4 AM) following the manufacturer's instruction. All experiments were carried out at room temperature using freshly isolated cells, attached to the poly-L-lysine coverslips of the perfusion chamber.