Circulating Progenitor Cells in Infarction Figure 8. Migration capacity of isolated leukocytes assessed using time of acquisition by FACS and counting beads. Blood was collected at TP2 (84–108 h) post-MI and subjected to Histopaque-1077 density gradient centrifugation. The buffy coat was collected and washed in PBS. Cells were suspended in EBM-2 medium at a density of 0.5–16107 cells/mL and loaded on 6-well tissue culture inserts (3-mm pore ThinCert; Greiner Bio One). Medium with or without chemoattractants (i.e. SDF or NGF) was added in the bottom part of the inserts. Following transwell migration in a humidified CO2 incubator for 18 hrs, the migrated and non-migrated cells number was evaluated by FACS analysis with the assistance of acquisition time or counting beads. As described in Materials and Methods, migration index (ratio of migrated cell fraction in the total cells) of each sample was then estimated using both the acquisition time-based and beads-based approaches (A and B). Migration index evaluated in the presence of counting beads (C). doi:10.1371/journal.pone.0030389.g008 Data analysis and statistics Data are shown as geometric mean with 95% confidence intervals. For the evaluation of migration capacity, following normalization of the data using FACS requisition time or counting beads, migrated cells (in the down part of the inserts) were divided by the sum of the migrated cells and non-migrated cells (that were left in the upper part of the inserts) and multiplied by 100 to give the migration percentage. Migration index is the value of migration percentage in a sample divided by that in the control, i.e., %Migration~NMigrated cells NMigrated cellszNNon-migrated cells ; Migration index~%MigrationðsampleÞ %MigrationðcontrolÞ: Statistical analyses were performed using regression modelling, taking account of the repeated measurements from the same patient. If the overall test for a difference between groups (F-test) was statistically significant (p,0.05) then paired comparisons between groups were made. Skewed data were transformed to the logarithmic scale for analysis and results are presented as geometric means with 95% confidence intervals. Model assumptions were checked and influential outlying values were excluded. Bland and Altman plots [13] were used to determine concordance between techniques of migration flow cytometric measurement with the limits of agreement defined as the mean of the difference 61.96 standard deviations. It was considered statistically significant when P value,0.05. Analyses were carried out using Stata 11.2 (StataCorp LP 4905 Lakeway Drive College Station, Texas. USA). PLoS ONE | www.plosone.org 10 January 2012 | Volume 7 | Issue 1 | e30389