Preparation and use of MitoPY1 for imaging hydrogen peroxide in mitochondria of live cells

Abstract

Mitochondria peroxy yellow 1 (MitoPY1) is a small-molecule fluorescent probe that selectively tracks to the mitochondria of live biological specimens and responds to local fluxes of hydrogen peroxide (H(2)O(2)) by a turn-on fluorescence enhancement. This bifunctional dye uses a triphenylphosphonium targeting group and a boronate-based molecular switch to selectively respond to H(2)O(2) over competing reactive oxygen species (ROS) within the mitochondria. MitoPY1 can be used to measure mitochondrial H(2)O(2) levels in both cell culture and tissue models. In this protocol, we describe the synthesis of MitoPY1 and how to use this chemical tool to visualize mitochondrial H(2)O(2) in live cells. The preparation of MitoPY1 is anticipated to take 7-10 d, and assays involving microscopy of cultured mammalian cells can be performed in 1-2 d.

Figure 1

Mitochondria Peroxy Yellow 1 (MitoPY1). (a) Scheme for the synthesis. (b) Scheme showing H₂O₂-mediated activation of MitoPY1.

Figure 2

Fluorescence turn-on response of 5 μM MitoPY1 to H₂O₂. Time points represent 0, 5, 15, 30, 45 and 60 min after the addition of 100 μM H₂O₂. Excitation at 503 nm.

Figure 3

Imaging mitochondrial H₂O₂ in live cells with MitoPY1. HeLa cells were loaded with 10 μM MitoPY1 in DPBS for 45 min at 37 °C. The medium was then exchanged for fresh DPBS containing 25 nM Mitotracker Deep Red and 1 μM Hoechst. After the addition of either H₂O (control) or 100 μM H₂O₂, the cells were incubated for 60 min at 37 °C. (a–d) Control cells were then imaged with MitoPY1 (a), Mitotracker Deep Red (b), overlay of MitoPY1 (green), Mitotracker Deep Red (red) and Hoechst (blue) (c), or overlay of bright-field and Hoechst (blue) (d). Scale bar, 40 μm. (e–h) H₂O₂-treated cells were imaged with MitoPY1 (e), Mitotracker Deep Red (f), overlay of MitoPY1 (green), Mitotracker Deep Red (red) and Hoechst (blue) (g), or overlay of bright-field and Hoechst (blue) (h). Scale bar, 40 μm. (i–l) The region of H₂O₂-treated cells denoted in e is enlarged, showing MitoPY1 (i), Mitotracker Deep Red (j), overlay of MitoPY1 (green), Mitotracker Deep Red (red) and Hoechst (blue) (k), or overlay of bright field and Hoechst (blue) (l). Scale bar, 10 μm.