and goat anti-mouse IgG HRP conjugated (Santa Cruz) were used as primary and secondary antibodies respectively. Antibody reactive bands were detected by enzyme - linked chemilumines- cence (Pierce) [25]. Examination of the role of DA D2 receptor antagonist on homing of exogenously transplanted MSCs to the wound bed Passage three MSC cultures were maintained at 50% confluence. 5-Bromo-29-deoxy-uridine (BrdU) labeling reagent (Roche Applied Science) was added into the culture medium (1:1000) 72 hours before transplantation. The BrdU labeled MSCs were then harvested by trypsinization and resuspended in normal saline for injection. On the first day after wounding, 106 live MSCs were injected into the tail vein of both control and DA D2 receptor antagonist treated back skin-injured mice after 2 hours of DA D2 receptor antagonist injection. Thereafter, on completion of treatment schedule on day 6, the skin from the wound area was collected and the number of BrdU labeled MSCs transplants were determined immunohistochemically by using BrdU labeling and detection kit (Roche Applied Science) [55]. Determination of the incorporation of exogenously transplanted MSCs into newly formed blood vessels in wound bed Lipophilic tracer (CM-Dil; Invitrogen Bioservices) was used to label MSCs [56–57]. The MSCs were trypsinized from their passage 3 cultures and incubated in the working solution of CM- Dil (15 mM CM-Dil solution in HBSS) for 5 minutes at 37uC and then for an additional 15 minutes at 4uC. After labeling, cells were washed twice with PBS and resuspended in ice-cold normal saline for injection. On day 1 after wounding, 106 live labeled MSCs were injected into both control and DA D2 receptor antagonist treated back skin-injured mice via tail vein, two hours after injection of DA D2 receptor antagonist. On day 6, the wound tissues were collected, washed in PBS and fixed in OCT compound at 220uC for 1 hour, and cryostat sections were cut from the mid-portion of the wounds with a cryomicrotome. Frozen sections were immunostained with anti-CD31 antibody (Rat anti- mouse; BD Pharmingen, USA) and FITC-conjugated secondary antibody (Goat anti-rat; Millipore, USA) or biotin conjugated secondary antibody (Rabbit anti-rat IgG; Millipore, MA) and then analyzed either to determine incorporation of CM-Dil labeled transplanted MSCs into the newly formed blood vessels in wound bed or microvessel density using ABC staining kit (Vector Laboratories) and Nova-Red substrate solution (Vector Laboratories) [25]. In vitro Migration assay of MSCs Migration assays were performed in transwell dishes (Corning Costar) 6.5 mm in diameter with 8 mm pore filters. The upper side of the transwell filter was coated for 1 hour at 37uC with 0.1% (wt/vol) bovine gelatin (Sigma-Aldrich) in PBS. After incubation with MSCs (56105 cells) at 37uC for 24 hrs, 10 ng/ml VEGF (ProSpec) was added to the bottom chamber. Migration observed in the presence of 30% FCS and with medium alone served as positive and negative controls respectively. After overnight incubation of the transwell chamber at 37uC, 5% CO2, the upper side of the filters was carefully washed with cold PBS, and cells remaining on the upper face of the filters were removed with a Dopamine and Mesenchymal Stem Cells cotton wool swab. Transwell filters were stained using Gill’s haematoxylin, cut out with a scalpel, and mounted onto glass slides, putting the lower face on the top. Each experiment was performed in triplicate. Data were expressed as numbers of total migrated cells per insert after subtraction of the basal migration observed in the presence of DMEM alone (without presence of any growth factor). We investigated the influence of DA (1 mM) on chemotactic activity of 10 ng/ml VEGF [58]. siRNA Transfection The MSCs were trypsinized from their 3rd passage culture and were plated in 6-well plates at a density of 26105 cells/well in 2 ml antibiotic free normal growth medium (Murine Mesenchymal Stem Cell Expansion Medium; Millipore, USA) and allowed to attach overnight. The cells were then transfected with Akt siRNA (mice). Transfection was performed using siRNA transfection medium and siRNA transfection reagent (Santa Cruz Biotechnol- ogy) as per the manufacturer’s instruction. Thereafter, following transfection, the cells were left to further grow for 48 hours and finally western blot was performed to confirm the silencing of the Akt using anti-Akt antibody (Santa Cruz Biotechnology) [59]. The concentration of siRNA was optimized to ensure that it did not affect cell viability. To evaluate the role of the Akt in the migratory activity of MSCs, in vitro migration assay of siRNA transfected (Akt-silenced) MSCs were undertaken in transwell dishes (Corning Costar, Cambridge, MA) against chemotactic activity of 10 ng/ml VEGF. Western blot analysis to evaluate VEGFR-2 and Akt phosphorylation in MSCs Anti-VEGFR-2, anti-p-VEGFR-2, anti-Akt and anti-p-Akt antibodies (Santa Cruz Biotechnology) were used as primary antibodies. Protein extracts from cells of various experimental groups were subjected to SDS-PAGE and then blotted onto PVDF membranes (Millipore, Bedford, MA). Protein loading was verified by stripping and reprobing the membrane with anti-VEGFR-2 and anti-Akt. Antibody-reactive bands were detected by enzyme- linked chemiluminescence (Pierce ECL, SuperSignal, Pierce Biotechnology) [59]. Actin polymerization assay Cells of various experimental groups were grown on glass coverslips and fixed in 37uC with 4% paraformaldehyde in PBS for 10 minutes. The cells were then incubated with 5 ml 0.15 M glycine solution for 5 minutes and permeabilized with a solution of 0.1% (v/v) Triton X-100 in PBS for 5 to 10 minutes. Finally, these cells were incubated with 1 mg/ml FITC-phalloidin (Sigma) staining solution for 40 minutes at room temperature [60]. Statistical analysis Data are means of at least 6 different experiments 6 SEM. Student’s t-test was used to analyze differences between groups. p value ,0.05 was considered statistically significant [22]. Author Contributions Conceived and designed the experiments: PSD SB. Performed the experiments: SS. Analyzed the data: SS PSD SB. Contributed reagents/ materials/analysis tools: PSD SB. Wrote the paper: SS PSD SB. Designed experiments: SS. Assisted in performing experiments: SB. PLoS ONE | www.plosone.org 10 February 2012 | Volume 7 | Issue 2 | e31682