Macrophages induce ADM of pancreatic acinar cells. (A) Freshly isolated, activated mouse primary macrophages and mouse primary acinar cells were co-cultured in 3D collagen explant culture. At day 5, ADM events per well were quantified by counting. (B) Raw 264.7 and freshly isolated mouse primary acinar cells were in vivo labeled with Vybrant dyes (pseudocolors: blue, macrophages [M]; red, acinar cells [AC]) and co-cultured in 3D collagen explant culture. At day 6, co-cultures were analyzed for duct (D) formation by fluorescence microscopy (bar, 50 µm). See also Fig. S2 A. (C) Primary pancreatic acinar cells were isolated and embedded in collagen in µ-Slides (Ibidi) for cell co-cultivation (magenta area in scheme). Similarly, Raw 264.7 cells (cyan areas in scheme) were embedded in collagen (or collagen alone as a control). Cells in the collagen matrix were overlayed with Waymouth media. ADM events in the acinar cell area were determined by counting ducts. (D) Primary mouse pancreatic acinar cells were isolated and cultivated in 3D collagen explant culture in presence of Raw 264.7–, WR19M.1- or primary mouse macrophage-conditioned media, and ADM events per well were determined (graph). (E) Photos show ducts obtained (at day 5) after treatment with Raw 264.7–conditioned media as compared with untreated acinar cells and cells treated with 50 ng/ml TGF-α. Bars, 100 µm. See also Fig. S2 B. (F) Primary mouse pancreatic acinar cells were isolated and treated with Raw 264.7–conditioned media to induce ADM. At day 2, cells were isolated from the 3D collagen explant culture, and quantitative real-time PCR for indicated markers of ADM events was performed. (G) Primary mouse pancreatic acinar cells were isolated and treated with Raw 264.7–conditioned media to induce ADM. Cells were isolated from the 3D collagen explant culture, and cell lysates were analyzed for expression of the ductal marker CK-19 or the acinar marker amylase by Western blotting. Silver staining served as a loading control. (H) Primary mouse pancreatic acinar cells were isolated and treated with Raw 264.7–conditioned media to induce ADM in presence of 3 µg/ml TGF-α neutralizing antibody (mTGFα-NAB) or 1 µM Erlotinib as indicated. Treatment with 50 ng/ml TGF-α served as a comparison to conditioned media. At day 5, ADM events per well were quantified by counting. The gray line represents onefold ADM events. In the experiments depicted in A, C, D, F, and H, bar graphs show means ± SD of n = 3 experiments. The asterisk indicates statistical significance as determined by the Student’s t test. All experiments depicted have been repeated with similar results at least three times.