Work

A Caspase-3 Reporter for Fluorescence Lifetime Imaging of Single-Cell Apoptosis

Year: 2018

Type: article

Abstract: Fluorescence lifetime imaging (FLIM) is a powerful imaging modality used to gather fluorescent reporter data independent of intracellular reporter intensity or imaging depth. We applied this technique to image real-time activation of a reporter for the proteolytic enzyme, caspase-3, in response to apoptotic cell death. This caspase-3 reporter activity provides valuable insight into cancer cell responsiveness to therapy and overall viability at a single-cell scale. Cleavage of a aspartate-glutamate-valine-aspartate (DEVD) linkage sequence alters Förster resonance energy transfer (FRET) within the reporter, affecting its lifetime. Cellular apoptosis was quantified in multiple environments ranging from 2D flat and 3D spheroid cell culture systems to in vivo murine mammary tumor xenografts. We evaluated cell-by-cell apoptotic responses to multiple pharmacological and genetic methods of interest involved in cancer cell death. Within this article, we describe methods for measuring caspase-3 activation at single-cell resolution in various complex environments using FLIM.

Source: Cells

DOI: 10.3390/cells7060057

Authors: Johanna M. Buschhaus, Brock Humphries, Kathryn E. Luker, Gary D. Luker

Institution: University of Michigan

Language: English

Topic: Advanced Fluorescence Microscopy Techniques

Subfield: Biophysics

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

SDG: Good health and well-being

Open Access status: gold

Estimated APC paid: $3,258