Cytoplasmic transfer evaluation by CFSE stain.

<p>Data are presented as an overlay of three different cytometric histograms (A). Empty histograms represent untreated cells at the outset. Grey and light grey histograms are generated using R1 and R2 gates (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035766#pone-0035766-g001" target="_blank">Fig. 1</a>) and therefore represent isolated and conjugated cells, respectively. The results show that MFI is higher in R2 gated events if compared to R1 gated events for each time points. CD4+ T cells staining with CFSE and DiI after 30 min of FasL treatment were analyzed by fluorescence deconvolution microscopy (B). The image shows a nanotube connecting two cells (thin arrow) and the uptake of red patches by green cells (thick arrows). Time-course of CFSE exchange (C) was evaluated for the indicated times of FasL treatment with different pharmacological agents, Lat A (Latrunculin A), Cyto B (Cytochalasin B), PP2 and Noco (Nocodazole). In particular, it is possible to observe a significant CFSE uptake from red cells, which was strongly inhibited by Lat A and Noco. Results from n = 6 separate experiments showed statistically significant differences for all inhibitors (p<0.05). Percentage of cell conjugates (D) was compared among all experimental conditions (FasL <i>vs</i> specific inhibitors). Statistical analyses of inhibitors effect on CFSE exchange (C) and cell conjugates formation (D) highlight a different behaviour of Lat A and Noco. Lat A treatment led a strong decrease of both phenomenon, whereas Noco treatment affected majorly cytoplasmic material transfer.