Protocol update for late endothelial progenitor cells identified by double-positive staining

Endothelial progenitor cells (EPCs), which are precursors of endothelial cells (ECs), have the capacity to circulate, proliferate and differentiate into mature ECs. EPCs are primarily identified by the uptake of 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine-labelled acetylated low-density lipoprotein (Dil-acLDL) and the binding of fluorescein-isothiocyanate (FITC)-conjugated Ulex europaeus agglutinin lectin (FITC-UEA-I). However, the cytoplasm and nucleus are usually stained by FITC-UEA-I via a typical method to double-stain late EPCs. It is necessary to explore a new method to improve the quality of fluorescence photomicrographs of late EPCs stained with FITC-UEA-I. Here, we described an updated protocol for double-staining late EPCs with Dil-acLDL and FITC-UEA-I, with the cells more optimally stained with FITC-UEA-I.