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Summary: Single-cell techniques have revolutionized biology; however, the required sample processing inherently implies the loss of spatial localization. Here, using an approach called photoconversion of areas to dissect micro-environments (PADME), we detail steps to isolate live single cells from a primary breast tumor while retaining spatial information by combining cell photolabeling and FACS (fluorescence-activated cell sorting). These live cells can be subsequently used for myriad techniques, from flow cytometry to single-cell RNA sequencing or other single cell “omics” approach.For complete details on the use and execution of this protocol, please refer to Baldominos et al. (2022). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.