Fig. 2.

CCL-1 selectively responds to Cu⁺ and can measure dynamic changes in Cu⁺ levels in living cells. Relative bioluminescence response of CCL-1 (5 μM, in 50 mM Tris, pH 7.4, 5 mM GSH) after 1 h incubation with (A) varying concentrations of Cu⁺ and (B) various biologically relevant s-block (1 mM) and d-block (100 μM) metal ions and cobalamin (vitamin B₁₂). Signals are integrated over 1 h and expressed as relative photon fluxes normalized to CCL-1 bioluminescence with no metal ion treatment. (C) Bioluminescent signals from PC3M-luc cells probed with CCL-1. Cells were supplemented with CuCl₂ for 24 h, followed by addition of CCL-1 (25 μM) ± the membrane-permeable copper chelator NS3′ (200 μM). Total photon flux was integrated over 2 h. Statistical analyses were performed with a two-tailed Student's t test. ****P ≤ 0.0001, and error bars are ±SD (n = 3). (D) Representative images of PC3M-luc cells treated with CuCl₂ and imaged with CCL-1. (E) Bioluminescent signals from PC3M-luc cells ± NS3′ (200 μM) and imaged with CCL-1 (50 μM). Total photon flux was integrated over 2 h. Statistical analyses were performed with a two-tailed Student's t test. **P ≤ 0.01, and error bars are ±SD (n = 3).