Zinquin Detection of Apoptosis[1]
This protocol is adapted from research data and provided for reference only. Adjustments may be necessary based on specific experimental requirements.
1. Reagent Preparation: Zinquin is dissolved in dimethyl sulfoxide (DMSO) at a concentration of 5mM and stored at -20°C. Before use, Zinquin is diluted to the working concentration (e.g., 25μM) with Hanks' Balanced Salt Solution (HBSS, pH=7.4, containing 1.3mM Ca²⁺, 0.9mM Mg²⁺, 4.2mM NaHCO₃). Other reagents include ZnSO₄·7H₂O, sodium pyrithione (a zinc ionophore), TPEN (a zinc chelator).
2. Cell Preparation: Cells (5×10⁶ cells/mL) are incubated at 37°C with 25μM ZnSO₄ and a specified concentration (typically 1–4μM) of sodium pyrithione for 40min, followed by three washes with HBSS to remove extracellular zinc. Alternatively, cells are incubated with different concentrations (0–100μM) of TPEN in culture medium for periods ranging from tens of minutes to several hours to reduce intracellular labile zinc levels.
3. Zinquin Labeling and Fluorescence Detection: Treated cells are incubated with Zinquin (25μM) in HBSS at 37°C for 30min. After incubation, cells are collected, and the fluorescence intensity of Zinquin is measured using a microplate reader (excitation/emission wavelengths = 368/490nm).
Precautions:
(1) Zinquin is specific for Zn²⁺ (Cd²⁺ can also enhance fluorescence, but its content in biological tissues is extremely low; Ca²⁺ and Mg²⁺ have almost no effect; Cu²⁺ and Fe²⁺ quench fluorescence). Zinquin mainly forms 1:1 and 1:2 complexes with Zn²⁺, with relatively low binding constants (approximately 10⁶L/mol), and may not detect zinc tightly bound to the active sites of metalloenzymes.
(2) The free acid form of Zinquin can passively cross membranes without requiring esterase hydrolysis. At high concentrations, Zinquin may transport zinc in a 1 Zn²⁺ exchange for 2 H⁺ manner, a process inhibited by transmembrane pH gradients.
(3) The fluorescence signal remains stable for 1–2 hours; washing after labeling does not affect fluorescence intensity, indicating that the probe is effectively trapped within the cells.
(4) For your safety and health, please wear a lab coat and disposable gloves during operation.
References:
[1] Zalewski PD, Forbes IJ, Betts WH. Correlation of apoptosis with change in intracellular labile Zn(II) using zinquin [(2-methyl-8-p-toluenesulphonamido-6-quinolyloxy)acetic acid], a new specific fluorescent probe for Zn(II). Biochem J. 1993 Dec 1;296 ( Pt 2)(Pt 2):403-8. |
