Circulating Progenitor Cells in Infarction Figure 1. SOP of the ProMIS. Left: Characterization of circulating progenitor cells (CPCs) and monocytes in peripheral blood of MI patients. Right: Evaluation of migration capacity of CPCs by flow cytometry. PB, peripheral blood; TP, time point; WBC, white blood cell; CPC, circulating progenitor cell; MNC, monocyte; RBC, red blood cell. doi:10.1371/journal.pone.0030389.g001 concentration of antibody was used. The signal from samples stained with KDR was always dim even when an excess of antibody was used. The optimal concentration of each individual antibody was not always as recommended by the vendor (Green Bar, Figure 6) thus implying the necessity of validation by the customer. The freshness and quality of the PB samples are relevant for the evaluation of CPCs Patients with MI refer to the clinic as emergencies at any time of day or night. Therefore, in order to maximise the opportunity for recruiting patients into a study like ProMIS, we investigated the effect of storing samples at 4uC for 24 hr before subjecting samples to immunostaining for flow cytometry analysis using data from 4 patients (24 samples in total; 4 patients63 replicates62 time points). As shown in Figure 7, it appeared that there is higher percentage of lymphocytes, but no change of the monocyte population after storage for 24 hrs (Figure 7A). Furthermore, the storage dramatically affected the quantification of distinct CPC populations with a CD34+, CD133+, KDR+ or CD117+ phenotype (Figure 7B–E). After mononuclear cells were isolated for functional tests such as migration assay, freshly processed samples appeared clear (Figure 7F), while clotting blocks were often observed from the tube containing a stored sample (Figure 7G). Clotting also occurred when venipuncture was technically difficult. These samples yielded artificially low levels of CPCs by flow cytometry. Evaluation of migration index using counting beads and acquisition time The migration assay is routinely used to estimate one fundamental functional property of mononuclear cells or isolated CPCs, e.g. the responsiveness to a chemoattractant. Flow cytometry can be subsequently applied to characterize distinct CPCs in the migrated and non-migrated fractions. However, it is not possible to quantify absolute cell numbers directly using the majority of flow cytometers such as a FACS Canto II. Therefore, following transwell migration of leukocytes toward SDF-1 or NGF, we compared the migration indexes which are normalized by using either the addition of counting beads or calculating acquisition time by the flow cytometer in 20 samples (10 patients62 replicates). When SDF-1 was used as chemoattractant, PLoS ONE | www.plosone.org 3 January 2012 | Volume 7 | Issue 1 | e30389