Methods. 2016 Oct 15;109:81-91. doi: 10.1016/j.ymeth.2016.07.001.

Exerting better control and specificity with singlet oxygen experiments in live mammalian cells.

Westberg M1, Bregnhøj M1, Banerjee C1, Blázquez-Castro A2, Breitenbach T1, Ogilby PR3.
1 Department of Chemistry, Aarhus University, Aarhus 8000, Denmark.
2 Department of Chemistry, Aarhus University, Aarhus 8000, Denmark; Aarhus Institute of Advanced Studies, Aarhus University, Aarhus 8000, Denmark.
3 Department of Chemistry, Aarhus University, Aarhus 8000, Denmark. Electronic address: progilby@chem.au.dk.

 

Abstract

Singlet molecular oxygen, O2(a1Δg), is a Reactive Oxygen Species, ROS, that acts as a signaling and/or perturbing agent in mammalian cells, influencing processes that range from cell proliferation to cell death. Although the importance of O2(a1Δg) in this regard is acknowledged, an understanding of the targets and mechanisms of O2(a1Δg) action is inadequate. Thus, methods that better facilitate studies of O2(a1Δg) in mammalian cells are highly desired. This is particularly important because, as a consequence of its chemistry in a cell, O2(a1Δg) can spawn the generation of other ROS (e.g., the hydroxyl radical) that, in turn, can have a unique influence on cell behavior and function. Therefore, exerting better control and specificity in O2(a1Δg) experiments ultimately reduces the number of variables in general studies to unravel the details of ROS-dependent cell dynamics. In this article, we summarize our recent efforts to produce O2(a1Δg) with increased control and selectivity in microscope-based single-cell experiments. The topics addressed include (1) two-photon excitation of a photosensitizer using a focused laser to create a spatially-localized volume of O2(a1Δg) with sub-cellular dimensions, (2) protein-encapsulated photosensitizers that can be localized in a specific cellular domain using genetic engineering, and (3) direct excitation of dissolved oxygen in sensitizer-free experiments to selectively produce O2(a1Δg) at the expense of other ROS. We also comment on our recent efforts to monitor O2(a1Δg) in cells and to monitor the cell’s response to O2(a1Δg).

KEYWORDS:

C11-BODIPY(581/591); Fluorescent ROS probes; Liperfluo; Optogenetics; Reactive Oxygen Species (ROS); Two-photon excitation