Quantitative analysis of Weibel-Palade bodies.

Weibel Palade bodies (WPBs) are vesicles found in endothelial cells which carry the multimeric protein von Willebrand factor (VWF). As cellular confluency has been shown to influence the number of WPBs in endothelial cells, we propose to test two methods of attaining endothelial cell confluence to inform on the relevancy of cellular culture methods when analyzing endothelial WPBs. We test these cellular culture methods in two endothelial cell types, human umbilical vein endothelial cells (HUVECs) and endothelial colony forming cells (ECFCs). One method maintains a constant incubation time of 96 hrs. while varying the seeding density. The second method maintains a constant seeding density of 30,000 cells/cm2 while varying incubation time. In comparing these two methods, we evaluate the nuclei count, total WPB count, and WPB/nuclei count for each. Our results show that there is a trend of increasing nuclei count, total WPB count, and WPB/nuclei count as incubation time and seeding density increases. However, there is no difference in WPB/nuclei quantification whether confluency is reached via a constant seeding density or a constant incubation time. In addition, we show that confluency plays a major role in WPB/nuclei generation as we demonstrate higher WPB/nuclei counts in confluent cultures compared to sub-confluent cultures.