Dopamine and Mesenchymal Stem Cells Figure 1. Treatment with dopamine (DA) D2 receptor specific antagonist significantly increased the number of circulating MSCs in peripheral blood of wound bearing mice. (A) Effect of eticlopride (DA D2 receptor specific antagonist) on the number of circulating MSCs in peripheral blood of wound bearing mice. Blood was collected from both the control and eticlopride treated wound bearing mice at different time intervals after injury, mixed with EDTA, treated with RBC lysis buffer and incubated with anti-CD45, anti-CD34 and anti-CD105 antibodies. Cells were initially gated to exclude dead cells and debris and then the CD342 CD452 cell population was selected and evaluated for presence of CD105, a specific surface marker of MSCs. Results are representative of six separate experiments each yielding similar results. (B) Graph showing that DA D2 receptor antagonist treatment have significant positive regulatory effect on MSC mobilization as significantly higher number of MSCs (CD342 CD452 CD105+ cells) was evident in peripheral blood of mice compared to vehicle treated controls at all time points after wounding. (*, P,0.05). (C) Characterization of MSCs by flow cytometry. Analysis with flow cytometer showed that over 95% cells of the total 3rd passage murine bone marrow cell populations were positive for both SSEA-4 and CD105 (specific surface markers of MSCs) and negative for CD34 and CD45, thus confirming their identity as MSCs. BM-MSCs were cultured in appropriate induction media to evaluate osteogenic and adipogenic differentiation potential of these stem cells and were confirmed by von Kossa staining (D) and Oil-red O staining (E) respectively. (D) To confirm osteogenic differentiation, mineralized deposits in the extracellular matrix were visualized by von Kossa staining. Differentiated cells were fixed with 4% paraformaldehyde, washed and then stained with 5% silver nitrate (Sigma) solution in the absence of light at room temperature for 30 minutes. Cells were then exposed to sunlight for 5 minutes and excess silver staining was removed by washing 2–3 times with a 5% sodium thiosulfate solution and ultimately washed with distilled water. (E) Adipogenic differentiation of cultured MSCs was confirmed by Oil-red O staining. Cells were fixed with 4% paraformaldehyde and washed PLoS ONE | www.plosone.org 3 February 2012 | Volume 7 | Issue 2 | e31682