Dopamine and Mesenchymal Stem Cells with 60% isopropanol. After completely drying the flasks Oil Red O working solution were added and stained for 15 minutes. Then all Oil Red O were removed and distilled water was added immediately to wash 2 times. Finally, the flasks were viewed under microscope and photographed. Original magnifications, 6200. doi:10.1371/journal.pone.0031682.g001 Several recent reports have indicated that VEGF is the predominant growth factor, which regulates wound angiogenesis [30–33] and che- motactic migration of MSCs both in vitro and in vivo [14,15,31,34–37], therefore further experiments were designed in vitro to determine the direct effect of VEGF on the migration of murine MSCs. The results showed that the numbers of migrated MSCs (CD342 CD452 CD105+ cells) in the presence of medium alone (DMEM without FCS) was very low and it increased in the presence of 30% FCS or VEGF (10 ng/ml) (Fig. 4A). This result corroborates well with the critical role of VEGF in regulating chemotaxis of MSCs into wound bed as reported by several other studies [15,31,34–37]. Therefore, further experiments were designed in vitro to evaluate the role of DA on VEGF induced migration of murine MSCs. Figure 2. Treatment with dopamine (DA) D2 receptor antagonist significantly accelerated mobilization of exogenously transplanted MSCs towards wound bed. (A) Flow cytometric analysis of DA D2 receptors in murine MSCs. To confirm that CD342 CD452 CD105+ SSEA-4+ cells express DA D2 receptors, in vitro expanded Linneg bone marrow cell population (containing CD452, CD11b2 cell populations) were initially gated to exclude dead cells and debris and then the CD342 cell population were selected and these CD342 CD452 cells were evaluated for presence of CD105, SSEA-4 and DA D2 receptors. Results showed that almost 86% cells of the total MSC population (both CD342 CD452 CD105+ cells and CD342 CD452 SSEA-4+ cells) express DA D2 receptors on their surfaces. (B) Western blot analysis of DA D2 receptors in murine BM-MSCs. H4 neuroglioma cell line and sarcoma-180 (S-180) tumor cells were used as positive and negative controls, respectively. (C) Effect of DA D2 receptor antagonist treatment on mobilization of exogenously transplanted MSCs towards wound site. MSCs were labeled with BrdU in culture and injected into the tail vein of both control and eticlopride treated back skin-injured mice. After completion of eticlopride treatment, at 6th day the skin from the wound area was collected and analyzed by immunohistochemistry using BrdU labeling and detection kit (Roche Applied Science). Significantly higher number of BrdU positive transplanted MSCs are located in the wound bed of eticlopride treated group than vehicle treated control group, showing DA D2 receptor antagonist treatment has significant positive effect on mobilization of exogenously transplanted MSCs towards wound site. Original magnifications, 6200. Results are representative of six separate experiments each yielding similar results. (D) Graphical representation showing significantly higher number of exogenously transplanted MSCs (BrdU positive cells) in wound bed of eticlopride treated groups compared to vehicle treated controls at day 6 post wounding (*, P,0.05). Number of transplanted MSCs cells was measured by counting the number of BrdU positive cells in 10 randomly chosen high power microscopic fields within the sections. doi:10.1371/journal.pone.0031682.g002 PLoS ONE | www.plosone.org 4 February 2012 | Volume 7 | Issue 2 | e31682