aphing the membrane through the microscope, and five random fields were taken. 3.6. Anchorage-Independent Growth Assay Soft agar colony-formation assays were performed as previously described with minor modifications. B16F10 cells in 1.5 mL of growth medium were mixed with 1.5 mL of 0.5% agarose in warmed growth medium containing vehicle or FA and layered on 0.5% base agar in 60-mm cell culture dishes. Culture medium containing scoparone was added only once; subsequently, medium without FA was added every week for 21 days until large colonies were evident. Cells were stained with crystal violet for colony counting. 3.7. Tube Formation Assay The tube formation assay was performed using 12-well plates coated with 100 L Matrigel basement membrane matrix per well and polymerized at 37 C for 30 min. HUVEC suspended in M199 medium containing 2% FBS were plated on the Matrigel at a density of 2 105 cells/well. FA was then added together with FGF1. After 6 h, The Matrigel-induced morphological changes were photographed, and the extent of capillary tube formation was evaluated by measuring the total tube length per 
field. 3.8. Rat Aortic Ring Assay The rat aortic ring assay was performed as described previously. In brief, 48-well plates were coated with 120 L of Matrigel per well and polymerized in an incubator. Aortas isolated from 6-week-old male Sprague-Dawley rats were cleaned of periadventitial fat and connective tissues in cold phosphate-buffered saline and cut into rings of 1~1.5 mm in circumference. The aortic rings were randomized into wells and sealed with a 100-L overlay of Matrigel. FGF1 in 500 L of serum-free M199 with or without FA added into the wells, and the fresh medium was exchanged for every 2 day. After 6 day, microvessel sprouting was fixed and photographed using an inverted microscope. 3.9. Chick Chorioallantoic Membrane PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19818716 Assay The chick chorioallantoic membrane assay was performed as described previously. 3.10.The wells were washed and blocked with 3% bovine serum albumin in PBS for 2 h. FA with 1% BSA in PBS was added with FGFR1 or FGFR2 to FGF1-coated wells. After 3 h of incubation, the wells were washed thrice with PBST. FGFR1 or FGFR2 bound to FGF1 was determined by biotinylated anti-human IgG and horseradish peroxidase-conjugated streptavidin, developed with tetramethylbenzidine substrate reagent and quantified by measuring the absorbance at 450 nm. 3.13. Matrix Metalloproteins Activity Assay The activity of MMP-9 and MMP-2 was determined by the QuickZyme MMPs activity assay according to the manufacturer’s instructions. Briefly, after treatment, cells were washed with fresh medium and replaced with serum-free medium. After an additional 24 h, the medium was collected and centrifuged at 10,000 g for 10 min. The respective supernatant was added to the 96-well strip coated with MMP-9 antibody or MMP-2 antibody and incubated at 4 C overnight. After washing with wash HC-067047 manufacturer buffer 3 times, 50 L assay buffer were added into the well, followed by adding 50 L detection reagent. After incubation at 37 C for 1 h, OD405 was measured with a Microplate Reader. 3.14. Immunofluorescence Analysis The effects of FA on FGF1-induced expression of FGFR1 phosphorylation in HUVEC were examined using an immunocytochemical method. Cells were pretreated with or without FA for 24 h in the presence of FGF1. For immunofluorescent labeling, anti-p-FGFR1Y154 antibody was used as the primary antibody and goat anti-rabbit IgG-FITC was used as a Int. J. Mol
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