Probe L

Operating Instructions (The following is a recommended experimental protocol for reference only, and adjustments are required based on specific requirements)

1. Stock Solution Preparation
1.1 Solvent: For most dyes, organic solvents are usually used for dissolution, such as anhydrous DMSO.
1.2 Concentration Recommendation: It is generally recommended to prepare a high-concentration stock solution at 1-10 mM.

2. Working Solution Preparation
2.1 Diluent: Optional diluents include 10.0 mM PBS buffer containing 1% DMSO (for ssDNA detection); DMEM cell culture medium (for cell imaging).
2.2 Working Concentration: The working concentration of Probe L is 10 μM; a concentration gradient of Zn²⁺ can be set according to experimental requirements (0-40 μM is used in literature, and 10 μM is used in cell experiments).
2.3 Notes: Adjust the working solution concentration as needed; prepare and use it immediately.

3. Staining Procedure
3.1 Adherent Cells: HeLa.
3.2 Incubation Conditions: First add Probe L (10 μM) and incubate at 37°C in the dark for 30 min; then add Zn²⁺ (10 μM) and continue incubation at 37°C in the dark for another 30 min before imaging.
3.3 Washing Step: After incubation, wash the cells 3 times with 10 mM PBS buffer to remove free probes and reduce background fluorescence.

4. Control Settings
4.1 Unstained Control: Used to adjust instrument voltage and eliminate autofluorescence of cells.
4.2 Negative Control: Incubate cells with only Probe L (10 μM) for 30 min without adding Zn²⁺.

5. Detection and Analysis
5.1 Instrument Type: Confocal fluorescence microscope; fluorescence spectrophotometer.
5.2 Wavelengths: Excitation wavelength is 365 nm; collect the emission channel at approximately 490 nm (green) for Zn²⁺ imaging, and collect the emission channel at approximately 550 nm for ssDNA detection.
5.3 Result Analysis: Almost no fluorescence is observed in cells when incubated with Probe L alone; bright green fluorescence appears in cells after adding Zn²⁺, indicating that Probe L has good cell membrane permeability and can achieve turn-on fluorescence imaging of Zn²⁺ in living cells. The fluorescence of the Probe L-Zn²⁺ complex at 550 nm is significantly enhanced after binding to ssDNA, which can be used for ssDNA detection.

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