Axiovert 40 cfl microscope

Cancer cells were suspended in type I collagen solution as described previously [41 (link)]. Briefly, collagen type I rat tail protein (Corning) was diluted to 2.2 mg/mL with DMEM, water, and NaOH and allowed to gel for 15 min at 37 °C. For morphologic examination of cells, cell colonies in three-dimensional collagen were examined using a Zeiss Axiovert 40 CFL microscope and pictures taken with a Nikon Coolpix 4500 camera.

The pancreas was removed, washed, minced into 1–2-mm pieces, and digested with solution containing collagenase I and soybean trypsin inhibitor at 37 °C for 30 min18 (link)43 (link). The collagen digestion was stopped by adding media containing 10% FBS. The digested pancreatic pieces were further washed and pipetted through 100-μm mesh to obtain pancreatic acinar cells. The freshly isolated pancreatic acinar cells were plated in Waymouth media solution containing neutralized collagen (2.2 mg/ml), and allowed to form 3D collagen gel over 15 min at 37 °C. The Waymouth media containing EGF (25 ng/mL) and FBS (2.5%) was then added on top of the gel. The media was replaced the next day and every other day with fresh Waymouth media containing EGF (25 ng/mL) and FBS (2.5%) that was supplemented with TGF-α (50 ng/ml). In additional experiments, the ROCK1/2 inhibitors Y276322 (10 μM) and Fasudil (25 μM), or the Rac1 inhibitor NSC23766 (20 μM) were added to the ex vivo cultures. After 4–5 days, the number of ‘duct-like’ structures developing in the acinar cells was counted using a Zeiss Axiovert 40 CFL microscope and pictures were taken with a Nikon Coolpix 4500 camera. The collagen gels were then fixed in formaldehyde, embedded in paraffin, and gel-sections stained to identify the ductal structures44 (link). The collagen gels were also processed for gene expression analysis by qRT-PCR.