Circulating Progenitor Cells in Infarction Figure 3. Effect fixative-containing RBC lysis buffer on cell yield. (A) Typical scatter plot of samples treated with fixative-containing lysis buffer. (B) Typical scatter plot of samples treated with fixative-free lysis buffer. (C) Effect of fixative in RBC lysis buffer on cell yield during FACS acquisition using counting beads compensation. (D) Effect of fixative in RBC lysis buffer on the percentage of lymphocyte and monocyte populations. Data are presented as geometric means with 95% confidence intervals. doi:10.1371/journal.pone.0030389.g003 based method, the migration of PB WBCs with response to SDF-1 or NGF was evaluated (Figure 8C). We demonstrated that the PB WBCs from MI patients migrated to SDF-1 but not NGF, which were indicated by one way ANOVA with LSD post hoc correction (P = 0.022 for SDF-1 and P = 0.695 for NGF, n = 28). Discussion This study describes several technical implementations for enumerating and characterising CPCs in the ProMIS trial. We first demonstrated that the lyse-wash step is superior to the lyse-no-wash step after immunostaining of peripheral blood. Using the lyse-and-wash technology, blood cell populations can be separated better and dead cells can be easily excluded either by a FSC-SSC plot or a fluorophores-conjugated viability dye. Furthermore, the sample solution prepared by a lyse-no-wash step is sticky and can block the fluid system of a cytometer like the BD Canto II. Samples need to be highly diluted with H2O when a lyse-no-wash method is applied, which results in extension of the running and analyse time of flow cytometry and thus becomes cost-inefficient. However, we acknowledge the appropriateness of the lyse-no-wash technique under specific circumstances; it is clear that the minimal manipulation of blood cells leads to less cell loss and less disturbance of fragile cells, which is particularly the case when one or two-colour markers (e.g. CD34) are of interest and it is not necessary obtain a large number of captured cell events (e.g. . = 16105 lymphocytes) [14,15]. However, when multicolour antigenic analysis is required and time/cost effects have to be considered, the lyse-wash technique may provide more advantages [16]. Next, we compared the effect of RBC lysis buffers with or without the fixative paraformaldehyde. The fixative-free RBC lysis PLoS ONE | www.plosone.org 5 January 2012 | Volume 7 | Issue 1 | e30389