Circulating Progenitor Cells in Infarction Figure 6. Titration of individual CPC markers before multicolour flow cytometry. Data are presented as geometric means with 95% confidence intervals. When without dilution, the volume of each antibody used for staining 100 m0 of blood is: 10 m0 of CD133-APC (50 CD133), 5 mL of CD34-PECy7 (50 mg/mL), 20 mL of KDR-FITC (50 mg/mL), 20 mL of CD164-PE (50 mg/mL), 5 mL of CD117-PE (50 mg/mL), and 20 mL of CD164-PE (25 mg/mL), respectively. The green bars show the dilution ratio of individual antibodies as recommended by vendors. The purple bars show the optimal dilution ratio of some antibodies according to our titration tests. doi:10.1371/journal.pone.0030389.g006 gations and treatment information are recorded in a case report form (CRF). Patients have a cardiac magnetic resonance (CMR) assessment on day 5 after presentation to determine the extent of MI injury (scar volume) and then 3 months later to assess the final volume of the scar. The protocol for this study was approved by the UK NHS Research Ethics Committee (REC reference number: 09/H0104/58). FACS analysis One hundred mL of peripheral blood samples collected in ethylenediaminetetraacetic acid (EDTA) containing tubes are incubated for 30 min at 4uC with mouse-anti-human monoclonal antibodies including kinase insert domain receptor (KDR) conju- gated with fluorescein isothiocyanate (FITC) (R&D Systems, Minneapolis, MN, USA), CD133/2 (clone 293C3) conjugated with allophycocyanin (APC) (Miltenyi Biotec, Bergisch Gladbach, Germany), CD34 (clone 8G12) conjugated with phycoerythrin- Cy7 (PE-Cy7) (BD Bioscience, San Jose, CA), CXCR4 (CD184) conjugated with PE (Invitrogen, Paisley, UK), CD164 (clone N6B6) conjugated with PE (Becton Dickinson, San Jose, CA), CD117/C- KIT (clone 104D2) conjugated with PE (Invitrogen, Paisley, UK), TrkA (Clone 165131) conjugated with PE (R&D Systems, Minneapolis, MN, USA), CD14 conjugated with FITC and CD16 conjugated with PE (Invitrogen, Paisley, UK) , respectively. Additional samples are stained with appropriate isotype control antibodies following the fluorescence-minus-one method. The isotype control antibodies we used were Mouse IgG1-FITC (Invitrogen, Paisley, UK), Mouse IgG1-PE (Invitrogen, Paisley, UK), Mouse IgG2b-APC (Miltenyi Biotec, Bergisch Gladbach, Germany) and Mouse IgG1 PE-Cy7 (BD Bioscience, San Jose, CA). The following steps are then carried out in sequence: N Cells are lysed with FACS lysis buffer (BD Bioscience, San Jose, CA) for the lyse/no wash procedure. N For the lyse/wash procedure, samples are subjected to lysis of red blood cells (RBC) for 15 min at room temperature using either 16 FACS lysis buffer (BD Bioscience, San Jose, CA) or high yield fixative free lysis buffer (Invitrogen, Paisley, UK). N Cells are spun down for 10 min at 3006g and then stained with the Fixable Viability Dye eFluorH 780 (eBioscience, San Diego, CA) for 30 min at 4uC. N Cells are washed with PBS and fixed with 1% paraformalde- hyde (PFA). AccuCheck counting beads are occasionally included for cell quantification according to the manufactur- er’s instructions (Invitrogen, Paisley, UK). N Flow cytometric acquisition is performed on a FACS Canto II system (BD Bioscience, San Jose, CA) within one week. FACS analysis is carried out using the FACS Diva 6.0 software (BD Bioscience, San Jose, CA). Migration assay Peripheral blood samples at TP2 in EDTA tubes are diluted with PBS supplemented with 2 mM EDTA and 0.5% BSA (v/v, PLoS ONE | www.plosone.org 8 January 2012 | Volume 7 | Issue 1 | e30389