# FRET detection and in situ quantification of efferocytosis using designed enzymatic activity

> **NIH NIH R01** · BAYLOR COLLEGE OF MEDICINE · 2024 · $331,200

## Abstract

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...

## Key facts

- **NIH application ID:** 10880620
- **Project number:** 5R01GM148812-03
- **Recipient organization:** BAYLOR COLLEGE OF MEDICINE
- **Principal Investigator:** VLADIMIR V DIDENKO
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $331,200
- **Award type:** 5
- **Project period:** 2022-09-21 → 2026-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10880620

## Citation

> US National Institutes of Health, RePORTER application 10880620, FRET detection and in situ quantification of efferocytosis using designed enzymatic activity (5R01GM148812-03). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10880620. Licensed CC0.

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