PS exposure and reconstitution experiments of cultured HUVECs.

<p>ECs were cultured in medium with normal serum (●, ○), uremic serum (●, ○), and normal serum with mixed uremic toxins (●, ○) for 24h, respectively (filled circles). Then the cells were washed and incubated with normal serum for another 24h (open circles). At indicated time points (0, 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48h), ECs were collected and incubated with lactadherin -Alexa Fluor 488 for 10 min in the dark before evaluation by flow cytometry. ECs cultured in normal serum for 48h were used as control. Each point represents mean ± SD for triplicate samples of independent experiments. <b>(A)</b> Kinetic mode of PS reversal on EC with different treatments. PS exposure on the outer membrane surface of EC occurred mainly from 12 to 24h when cultured with uremic serum or mixed toxins. After washing ECs, about 80% of PS reverted to the inner leaflet during the first 8h of culture with normal serum. ECs cultured for 24 h with normal or uremic serum were stained with CD31-Alexa Fluor 647 and PS exposure was detected using lactadherin-Alexa Fluor 488 and visualized using confocal microscopy. <b>(B)</b> Almost no lactadherin staining was observed on ECs cultured in normal serum. <b>(C)</b> Treatment of ECs with uremic serum led to retraction of cell margins, extension of filopods, and lactadherin (green) binding on filopods. The inset bar equals 10 μm.