Circulating Progenitor Cells in Infarction Figure 5. Fluorescence minus One (FMO) controls for the quantification of rare CPC events by FACS. Isotype controls were prepared by the combination of all the individual fluorophore-conjugated isotype control antibodies (A, B and C). FMO controls were prepared like isotype controls without adding a particular fluorophore-conjugated isotype control antibody, for instance fluorescence minus PE (D), fluorescence minus APC (E), fluorescence minus PE-Cy7 (F), respectively. The gating for each channel was defined to exclude nearly all background from the FMO controls on the 2D plots of FSC-H vs. individual fluorescence (e.g. PE, APC or PE-Cy7). Certain cell populations including CD117+ (G), CD133+ (H) and CD34+ (I) from a single sample were enumerated according to the gating strategy as above. doi:10.1371/journal.pone.0030389.g005 Materials and Methods Study design We selected four cohorts for assessment in ProMIS, namely patients presenting with either ST elevation MI (STEMI) or non- ST-elevation MI (NSTEMI), with or without type 2 diabetes mellitus. We are recruiting 18 patients prospectively for each cohort, thus 72 patients in total. Eligibility criteria are as follows: patients presenting with clinical features of STEMI or NSTEMI within 24 hours of symptom onset, aged 40 to 75 yrs on admission, and resident within 40 miles of the study centre. Patients are excluded if they have anaemia (haemoglobin, 10 mg/dL), cardiogenic shock, significant renal impairment 2 (eGFR,50 mL /min per 1.73 m ), haemodynamic instability, contraindications to undergoing a magnetic resonance scan (e.g. metallic implant, pacemakers, screws, claustrophobia etc), have had previous coronary event within the last 12 weeks, are participating in another clinical trial or show heightened anxiety during recruitment. Written informed consent is obtained directly from each eligible patient before the trial. 10 mL blood sample (TP1 sample) is then obtained within 12–24 hours of the onset of symptoms for FACS analysis. A second 50 mL blood sample (TP2 sample) is obtained at 84–108 hours after the onset of symptoms for FACS analysis and transwell migration assay of CPCs. Blood samples are transferred to the laboratory at 4uC and processed within 24 hours unless described differently. Demographic data and clinical data including past medical history, medication history, acute investi- PLoS ONE | www.plosone.org 7 January 2012 | Volume 7 | Issue 1 | e30389