Abstract Efferocytosis - the engulfment of dying cells is an essential regulatory process. It can either inhibit or promote self-immunization, inflammation and the release of viral or tumor DNA. Efferocytosis is a collective cellular event done by either professional phagocytes or by the surrounding tissue cells. The earlier view of apoptosis as a universally anti-inflammatory process has changed. It is now divided into the immunogenic and the tolerogenic (non-inflammatory) forms based the engulfment process. Therefore it became essential to have a broad-spectrum technology which could detect and quantify efferocytic processing in all participating cell types, However, currently there is no such assay. In this project we set out to overcome this limitation. This project will introduce a principally new technology detecting efferocytosis in tissue sections and in live cells. Our method isolates the unique enzymatic activity, present only transiently in the catalytic cycle of topoisomerases, and uses it as a basis for the new type of molecular labeling. The assay will use catalytic intermediates of the topoisomerization reaction as novel fluorescent probes. These FRET probes are capable of detecting and quantifying the essential marker of efferocytosis, nucleolytic activity of DNase II digesting DNA of apoptotic cells. The technology will selectively label and quantify apoptotic DNA disassembly in efferosomes, i.e. phagolysosomes of the cells that engulfed apoptotic nuclei. The new method will permit highly specific detection and the near instantaneous FRET quantification of efferocytic and phagocytic reactions in live and fixed cells and tissues. The proposal will reach these Specific Aims: 1. To develop the first in situ assay using catalytic intermediates of topoisomerization reaction as novel fluorescent probes detecting and quantifying efferocytosis in the tissue section format. To optimize specificity, sensitivity and reproducibility of the assay using in vitro and in situ models. To validate the new method in several models of efferocytic clearance using tissue sections. 2. To develop the first FRET assay technology using catalytic intermediates of topoisomerization reaction as novel FRET probes for labeling and quantification of efferocytosis in live cells at the level of individual cells. To optimize specificity, sensitivity and quantitative ability of the assay using in vitro, in situ models, and live cells. To validate the new FRET method in several models of efferocytic clearance using cultured cells. 3. To enable the subcellular assessment of efferocytosis efficiency by radically increasing sensitivity of the technology with help of novel advanced fluorescent tags - Ndots. To optimize the assay to achieve single molecule resolution in assessing its molecular targets and in broad range signal quantification. To finalize development of the technology by validating its quantification capacity in several models combining apoptosis and it...