Evaluation of intra-cellular ROS during internalization and subsequent removal of SPIOn.

<p><b>a</b> hAFCs and <b>b</b> hCVCs were plated and labeled with DAF-FM to visualize intra-cellular ROS, before being incubated with SPIOn (35 or 100 µg/ml). ROS measurement was performed every hour (from 1^st to 5^th) during SPIOn internalization (see experimental scheme). Data are expressed as ROS fluorescent signal normalized for Digitonin-PI. Unlabeled cells represent control condition (CTR). No statistically significant differences were observed between CTR and SPIOn labeled cells. ***p<0.001 versus CTR; °°°p<0.001 versus SPIOn (35 µg/ml). Two way analysis of variance (ANOVA); <b>c</b> hAFCs and <b>d</b> hCVCs were plated and labeled with DAF-FM and then were incubated with SPIOn (35 µg/ml). ROS evaluation was done at different time (6, 12, 24, 48 and 72 hours) after SPIOn addition. Data are expressed as ROS fluorescent signal normalized for Digitonin-PI. Unlabeled cells represent control condition (CTR). ***p<0.001 versus respective 6 hours; °°°p<0.001 respective 12 hours; +++p<0.001 versus respective 24 hours. Two way analysis of variance (ANOVA) as specified in the Statistical section. To better visualize the significant difference in intra-cellular ROS formation between hAFCs and hCVCs, we compared the two cellular groups at <b>e</b> 6, <b>f</b> 12, <b>g</b> 24, <b>h</b> 48 and <b>i</b> 72 hours after SPIOn removal. Data are expressed as ROS fluorescent signal normalized for Digitonin-PI. Unlabeled cells represent control condition (CTR). **p<0.01 and ***p<0.001 versus hAFCs. Two way analysis of variance (ANOVA), as specified in the statistical section.