Circulating Progenitor Cells in Infarction Figure 2. Comparison of lyse-wash and lyse-no-wash procedures. Whole blood samples were stained with progenitor cell markers and eFluor 780 fixable viability dye. Following RBC lysis with FCS lysis buffer (BD Bioscience), stained samples were washed with PBS (A, C and E) or stored at 4uC for subsequent FACS analysis within 5 days (B, D and F). doi:10.1371/journal.pone.0030389.g002 the Bland-Altman plot was shown in Figure 8A with the 95% limits of agreement between two techniques ranging from 20.27 to +0.40. When NGF was used as a chemoattractant, the Bland- Altman plot was shown in Figure 8B with the 95% limits of agreement between the two techniques ranging from 20.30 to +0.52 (Figure 8B). These data indicate that both techniques can be used to evaluate migration activity of CPCs to SDF-1 or NGF- 1. However, the acquisition method is based on the assumption of consistent speed during data collection, which is difficult for normalization because approximately just 11.59% of cells migrated to SDF (95% CI of geometric mean, 9.69–13.86%, n = 28), and the flow cytometric speed should be adjusted to allow detection accuracy for the non-migrated samples while reaching maximal acquisition for the migrated samples. Therefore, it may be more reliable to incorporate counting beads in order to precisely calculate the number of cells. Using the counting-bead PLoS ONE | www.plosone.org 4 January 2012 | Volume 7 | Issue 1 | e30389