Fig 1: Neutrophil transmigration from whole blood was analyzed after 4 h incubation. (a) The mean intensity SSC-A (granularity) of all transmigrated cells was significant different between fresh and stored blood with and without IL8 treatment. It revealed higher granularity of transmigrated cells, indicating higher activation of neutrophils after 24 h of blood storage as also confirmed by ROS production, shown in Figure 5. (b) The gating strategy for the analysis of transmigrated cells by flow cytometry is presented and neutrophils were identified based on FSC-A and SSC-A. (c) Neutrophils from stored blood transmigrated less than neutrophils from fresh blood. The IL8 stimulated transmigration was only significantly increased in stored blood. (d) Observing the whole transmigrated cellular population, the percentage of neutrophils is significantly higher in stored blood. Data were analyzed with one-tailed paired Student’s t-test (all assays were conducted in triplicates, and the mean is presented from n = 3 independent runs) and presented with mean ± SD (* p < 0.05, ** p < 0.01).