Mito-Cu(II)

Mito-Cu (II) exhibits high selectivity, rapid Cu²⁺ responsiveness, low cytotoxicity, excellent photostability, and stable fluorescence within the pH range of 4.0-11.0^[1].
Mito-Cu (II) (0-50 μM; 24 h) exhibits low cytotoxicity against HepG2 cells (TCHu 72), with cell viability exceeding 90% following incubation at 37 °C for 24 h at concentrations ranging from 0 to 50 μM^[1].
Mito-Cu (II) exhibits excellent photostability upon continuous laser irradiation in living HepG2 cells (TCHu 72)^[1].
Guidelines (The following is our recommended protocol, which serves only as a guide and should be modified according to your specific needs).
1. Preparation of Working Solution
Stock solution: Probe L (dissolved in DMSO to prepare a 1 mM stock solution, stored away from light).
Working solution: Dilute with cell culture medium (such as DMEM) to a final concentration of 10 μM.
Other reagents:
Cu (NO₃)₂ solution (exogenous Cu²⁺, generally added at 1 equivalent relative to the probe concentration or as required by experiments).
EDTA solution (chelating agent, used to restore fluorescence and verify reversibility).
Mitochondrial tracking dye: Mito-Tracker Deep Red (HY-D1783) (Ex/Em ≈ 644/665 nm, used for colocalization verification).
2. Experimental Procedures
2.1 Cell seeding: Seed HepG2 cells (such as TCHu 72) into a 24-well glass-bottom culture plate at a density of approximately 10,000 cells/well, and culture at 37°C with 5% CO₂ for 72-96 h until the cells reach approximately 90% confluence.
2.2 Probe incubation (staining):
Aspirate the original medium, and add fresh DMEM medium containing 10 μM Probe L.
Incubate in a 37°C, 5% CO₂ incubator for 15 minutes.
2.3 Washing: Aspirate the probe solution, wash the cells 3 times with PBS to remove unbound probe, and replace with fresh DMEM for imaging.
2.4 Basic imaging (mitochondrial targeting verification):
Place the culture dish directly into the temperature- and CO₂-controlled chamber of a confocal microscope.
Excitation (λex): 405 nm
Emission (λem): Collect signals from the blue/cyan channel (corresponding to the ~450 nm emission of free Probe L).
Co-staining with Mito-Tracker Deep Red (Ex 644 nm / Em 665 nm) can be performed simultaneously: after incubation and washing with Probe L, incubate the cells with 10 μM Mito-Tracker for 5-30 minutes, wash with PBS, then perform confocal imaging and calculate the Pearson correlation coefficient.
2.5 Real-time Cu²⁺ detection (On-Off-On dynamics):
First perform time-lapse baseline imaging of the mitochondrial region of cells stained only with Probe L (Ex = 405 nm).
Add Cu²⁺ solution directly into the culture dish (e.g., 1 equivalent of Cu (NO₃)₂), and continue time-lapse imaging. Observe that the fluorescence intensity in mitochondria gradually decreases, reaching the lowest value after approximately 20 minutes.
Add EDTA solution (1 equivalent) and continue imaging. Observe the gradual recovery of fluorescence (On), confirming reversible binding.
(Optional: Record a video throughout the process to document the real-time fluorescence switching process).

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.