Cd49f pe clone ebiogoh3

Prostate lobes were dissociated and stained for FACS based on published methods^{20 (link)}. Briefly, prostate lobes were dissected, minced, and incubated at 37°C in 10%FBS/DMEM containing 1 mg/ml collagenase (Gibco, cat. no. 17018–029). Further dissociation to a single cell suspension was achieved by incubation in Trypsin/0.05% EDTA (Invitrogen cat. no. 25300) for 5 minutes at 37°C, followed by serially passing through 18-G and 20-G needles and filtering through a nylon mesh filter with a 40 μm pore size. Cells were stained for 20 minutes at 4°C. Antibodies used are: Sca-1-APC (clone D7; eBioscience, cat. no. 17–5981–82), 1:500; Ter119-FITC (clone TER-119; eBioscience, cat. no. 11–5921–85), 1:250; CD31-FITC (clone 390; eBioscience, cat. no. 11–0311–85), 1:250; CD45-FITC (clone 30-F11; eBioscience, cat. no. 11–0451–85), 1:250; CD49f-PE (clone eBioGoH3; eBioscience, cat. no. 12–0495–83) 1:333. Cells were sorted using a FACS AriaII cytometer (BD Biosciences), and analysis of flow cytometry data was performed using FlowJo Software (Treestar). Following FACS, isolated cells were centrifuged at 500g for 5 minutes. The pellet washed in PBS, homogenized in lysis buffer from the RNAqueous-Micro RNA Isolation Kit (Ambion AM1931), the mirVana miRNA Isolation Kit (Ambion AM1560) and RNA extracted following kit instructions.