Mouse rat hgf quantikine elisa kit

FACS-isolated stromal cells were cultured in 10% FBS/DMEM in a 48-well tissue culture plate. When the cells were 80% confluent, old media was removed and cells were incubated in 150 μl 10% FBS/DMEM containing 10 μM purmorphamine (Calbiochem 540220) or DMSO control. In separate experiments, cultured stromal cells were treated with 10% FBS/DMEM containing 5 μg/ml recombinant Ihh protein (R&D 1705-HH-025) in 0.1% BSA or a BSA only control. In separate experiments, cells were transfected with miRNA mimics for miR-26a (Ambion, Assay MC10249) or miR-26b (Ambion, Assay MC12899) or a negative control (Ambion 4464076). After 22 h, media was removed and centrifuged at 10,000g for 5 min. To measure the concentration of Hgf in the media, 10 μl of the supernatant was used in each well of the Mouse/Rat HGF Quantikine ELISA Kit (R&D Systems, MHG00) and the plate processed according to manufacturer’s instructions. Each sample was analyzed in duplicate.

Individual prostate lobes were dissected in PBS, washed, and homogenized in PBS using a pestle. An equal volume of Lysis Buffer 2 (R&D 895347) was added and the homogenate incubated at room temperature for 30 min with agitation. The lysate was centrifuged at 10,000g for 30 min at 4°C, and the supernatant removed. Protein levels in each extract was determined using the Quick Start Bradford 1x Dye Reagent (Bio-Rad 500–0205), equal amounts of total protein were loaded into each well of the Mouse/Rat HGF Quantikine ELISA Kit (R&D Systems, MHG00) and the plate processed according to manufacturer’s instructions. Each sample was analyzed in duplicate, and Hgf levels for each sample was normalized to total protein in each well.