Circulating Progenitor Cells in Infarction Figure 7. Effect of blood storage time on CPC profiling. Peripheral blood samples from MI patients were processed either directly (,1 hr) or stored at 4uC for 24 hr before analysis. (A) Bar graph showing the percentage of lymphocyte and monocyte cell populations. (B, C, D and E) Bar graph showing the percentage of CD34-PE-Cy7, CD133-APC, KDR-FITC and CD117-PE in lymphocytes, respectively before and after storage for 24 hrs. (C and D) Representative images show that there was excessive clotting in the Leucosep tubes after leukocyte isolation when samples were stored for 24 h at 4uC. Data are presented as geometric means with 95% confidence intervals. doi:10.1371/journal.pone.0030389.g007 1:1); 30 mL of diluted blood solution is then loaded in a 50-mL LeucoSep tubes (Greiner, Frickenhausen, Germany) with 15 mL of Histopaque 1077 (Sigma, Poole, UK) and centrifuged at 8506g for 25 min. The buffy coat layers containing white blood cells are collected and washed twice with sterile PBS. Cells are then resuspended in EBM-2 (Lonza, Berkshire, UK) supplemented with 0.1% BSA. 0.5–16107 cells/mL of cells are loaded onto the upper part of ThinCertTM tissue culture polystyrene inserts (452.4 mm2 culture surface, 3.0 mm pore size; Greiner BioOne, Gloucester, UK) which are pre-assembled on 6-well plates containing medium as above with or without 100 ng/mL of recombinant human SDF-1a or b-NGF (PeproTech, Rocky Hill, NJ), respectively. Cells are then incubated at 37uC in a humidified incubator with 5% CO2 for 18 hrs. Cells from both parts of the transwell inserts are dissociated with Accutase (Invitrogen, Paisley, UK) and collected into FACS tubes. Following washing using PBS, migrated and non-migrated cells are stained with KDR-FITC, CD133/2 (293C3)-APC, CD34 (8G12)-PE-Cy7 and CXCR4 (CD184)-PE antibodies for flow cytometry analysis as described above. PLoS ONE | www.plosone.org 9 January 2012 | Volume 7 | Issue 1 | e30389