Rom the plasmatic concentration (38.01 ?34.23 pg/ml) when compared with PRP2-Ca (118.39 ?82.39 pg/ml) and PRP2-Thr (96.76 ?49.60 pg/ml) ?we considerAmable et al. Stem Cell Research Therapy 2013, 4:67 http://stemcellres.com/content/4/3/Page PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/14960617 11 ofFigure 6 Proinflammatory cytokine concentration. Concentrations of (A) IL-8, (B) IL-17, PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/12027669 and (C) TNF. *Statistically significant differences from the plasmatic concentration (analysis of variance followed by Dunnett's multiple comparison test, n = 6, = 0.05). PPP, platelet-poor plasma; PRP1, platelet-rich plasma after the first blood centrifugation step; PRP2, platelet-rich plasma after the second centrifugation step; PRP2-Ca, calcium-activated PRP2; PRP2-Thr, calcium plus human thrombin-activated PRP2.these values indicative of an increase and they should therefore be further studied. Eotaxin and MCP-1 showed significantly reduced concentration in activated PRP fractions; the eotaxin concentration dropped from a plasmatic concentration of 50.62 ?16.95 pg/ml to 23.75 ?8.52 pg/ml in PRP2-Ca and 22.19 ?4.76 pg/ml in PRP2-Thr, and the MCP-1 concentration from 245.59 ?187.52 pg/ml to 69.41 ?43.27 pg/ml in PRP2-Ca and 78.97 ?22.20 pg/ml in PRP2-Thr. The reduction may be caused by degradation of chemokines by the products secreted following degranulation, or their association with secreted molecules, and these issues should also be further studied. For chemokines, only one publication for RANTES was found, reporting a plasmatic concentration of 100 ?20 ng/mL and activated PRP concentration of 300 ?100 ng/mL [27]. Our research resulted in plasmatic RANTES concentrations of 2.76 ?1.97 ng/mL. Judging by the PRP composition described here, our findings support the use of PRP in wound healing and tissue regeneration, as high concentrations of PDGF and TGF are secreted from platelet -granules after activation. We also showed that growth factors are highly retained inside clots so, as already demonstrated by other authors, they would be slowly released in vivo [54,55]. These growth factors were also described as mitogenic and attractant for mesenchymal stem cells, which may continue secreting growth factors and may mediate regenerative effects during longer periods of time [56]. Our PRP preparation should now be tested in vitro in cell cultures in order to reveal its molecular effects on different cell types and in clinical trials in order to evaluate its in vivo effects in different pathologies.Conclusions Our study optimized a procedure for preparing PRP from human blood, 2-Chloro-3-methoxyaniline recovering more than 50 of the initial platelets with a low amount of other blood cells. This preparation was activated and different fractions collected along the procedure were analyzed in order to determine the growth factor content. Out of 37 proteins quantified, 12 were shown to be increased only after platelet activation.Amable et al. Stem Cell Research Therapy 2013, 4:67 http://stemcellres.com/content/4/3/Page 12 ofAbbreviations bFGF: Basic fibroblast growth factor; EGF: N-BOC-3-Fluoro-D-phenylalanine Endothelial growth factor; ELISA: Enzyme-linked immunosorbent assay; GM-CSF: Granulocyte acrophage colony-stimulating factor; G-CSF: Granulocyte colony-stimulating ijms17122034 factor; HGF: Hepatocyte growth factor; IFN: Interferon; IGF-1: Insulin-like growth factor-1; IL: Interleukin; IP-10: protein 10; MCP-1: Monocyte chemoattractant protein-1; MIP: Macrophage inflammatory protein; PDGF: Platelet-derived growth factor; PPP: Platelet-poor plasma; PRP: Platelet-rich plasma;.