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Summary: Currently, there is no protocol for growing and culturing primary pulmonary arterial cells (PACs) available from the Pulmonary Hypertension Breakthrough Initiative (PHBI). Here, we present a protocol for cultivating and maintaining three major PACs collected from patients with pulmonary arterial hypertension (PAH): endothelial (PAH-ECs), smooth muscle (PAH-SMCs), and adventitial cells (PAH-ADCs). We describe steps for obtaining PACs from PHBI, evaluating the growth of cells labeled with quantum dots (QDs), and staining endothelial cell (EC) markers for immunofluorescence imaging.For complete details on the use and execution of this protocol, please refer to Al-Hilal et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.