Specific determination of endothelial cell viability in the whole cell fraction from cryopreserved canine femoral veins using flow cytometry

An efficient method for specifically determining the viability of endotheli al cells (EC) from cells dissociated from the human saphenous vein was inve stigated. Three different methods, trypan blue staining assay, [H-3]-prolin e incorporation assay, and flow cytometry (FCM), combined with the fluoresc ein isothiocyanate conjugated with Griffonia simplicifolia agglutins (GS1-F ITC)/propidium iodide (PI) double staining, were used. Both trypan blue sta ining and [H-3] proline incorporation assays demonstrated less sensitivity to determine viability of EC differentially from the other cells. FITC-GS1 showed prominent binding to the vascular EC and could be counted by FCM inc luding PI on dead cells. Following the cryopreservation process, the GS1-FI TC/PI FCM analytical method was adopted to test simultaneously the viabilit y of whole cells and EC from the same tissue, human saphenous veins, and mo ngrel dogs' femoral veins after harvesting, antibiotic solution treatment, and thawing. The viability of the whole cells from veins decreased with a s ignificant difference (p < 0.05) from that of EC after thawing.