Pe cy7 conjugated anti cd45

Single cell suspensions of 3 week old pancreata were generated with a modified version of a protocol established by Sugiyama and colleagues, 2007 [26] (link). Briefly, pancreatic lymph nodes were removed, and pancreata were minced and sequentially incubated with collagenase D (1 mg/ml in Hanks buffered saline solution, Roche), trypsin (0.05%, Invitrogen), and dispase (2 U/ml, Invitrogen). Dnase1 (100 ug/ml, Sigma) was added during all enzyme incubations. Cells were washed with PBS between the collagenase and trypsin steps, and with FACs buffer (2% FBS, 10 mM EGTA, in PBS) between the trypsin and dispase steps. Suspensions were then filtered through 40 uM mesh, subjected to FC block (1∶200), and incubated with PE-conjugated anti-Cd49f (1∶50, Ebioscience), PE-CY7-conjugated anti-CD45 (1∶500, Ebioscience), and biotin conjugated anti-CD133 antibodies (1∶50, Ebioscience). Streptavidin conjugated to APC was then applied to detect CD133 positive cells (1∶100, Ebisocience). Cells were washed with FACs buffer between block, primary and secondary antibody incubations. DAPI (300 nM) was used as a live cell marker. Cell sorting was performed on a FacsARIAII (Becton Dickinson).