A step-by-step protocol for isolation and flow cytometry analysis of porcine cardiac valve leaflets

Flow cytometry analysis of GFP-expressing porcine valves is an indispensable model for studying partial heart transplantation immunobiology. Here, we present a protocol for the systematic isolation and phenotypic characterization of leaflet-derived cells from pulmonary and aortic valves. We describe steps for valve procurement, enzymatic digestion to single-cell suspension, preparation of samples with antibody, and viability staining and gating strategies for accurate and reproducible flow cytometric analysis. This approach provides valuable insight into valve biology and supports rigorous evaluation of cellular dynamics in experimental transplantation models.