Single Nuclei Isolation from Coronary Endarterectomy Tissue of Coronary Artery Bypass Graft Patients

Atherosclerosis is a chronic, progressive inflammatory condition characterized by the accumulation of plaque in the walls of arteries. During the progression of plaque formation, certain lesions become unstable, referred to as high-risk plaques, and have the potential to cause myocardial infarction (MI). An atherosclerotic plaque comprises smooth muscle cells, endothelial cells, immune cells, fat cells, and necrotic cells, any of which can cause plaque rupture. It is crucial to study cellular heterogeneity within atherosclerotic plaques to better understand the mechanisms underlying plaque instability. We present an easily reproducible protocol for isolating and validating nuclei from human coronary endarterectomy tissue obtained from patients undergoing coronary artery bypass graft (CABG) surgery. All tissues are kept on wet ice in Roswell Park Memorial Institute (RPMI) medium to preserve cellular integrity before harvesting. The tissue samples are finely minced and then gently disrupted with a micro pestle in lysis buffer to lyse cells and release nuclei. The isolated nuclei are then assessed for integrity and yield using the trypan blue exclusion assay in a compound inverted microscope. DAPI staining enables visualization of intact nuclei by confocal microscopy, and flow cytometry provides fluorescence-based quantification to assess nuclear isolation yield and validate the nuclear population. In contrast to traditional methods of isolation via single-cell dissociation, nuclear isolation reduces cellular stress and maintains the native transcriptomic environment, thereby preventing changes in gene expression. The high-quality nuclear preparation obtained through this method is compatible with downstream applications such as single-nuclei RNA sequencing (snRNA-seq).