WSP-5 (Synonyms: Washington State Probe-5)

This plan only provides a guide, please modify it to meet your specific needs.

1.Solution Preparation:

(1) Stock Solution Preparation: Equilibrate solid WSP-5 to room temperature and dissolve it in DMSO to prepare a stock solution with a concentration of 10 mM. Note: Unused stock solution should be aliquoted and stored in the dark at -20°C or -80°C to avoid repeated freeze-thaw cycles.

(2) Working Solution Preparation: Dilute the stock solution with an appropriate buffer (e.g., serum-free culture medium or PBS) to the desired working concentration, such as 10-100 μM. Note: Optimal working concentrations should be adjusted based on experimental conditions or consulted literature for gradient concentration settings. Working solutions must be freshly prepared.

2.Detection of Cellular H₂S Using WSP-5 ^[1] (Reference Only)

(1) Cell Treatment: Culture 2.5×10^5 4T1 cancer cells in a 24-well plate and allow them to adhere for 12 hours.

(2) WSP-5 Incubation: Incubate the cells with 100 μM WPS-5 probe for 30 minutes.

(3) Na₂S Treatment:Treat the cells with 200 μM Na₂S.

(4) Fluorescence Detection:After 10 minutes, acquire fluorescence images using confocal laser scanning microscopy (CLSM). λ ex/em = 502/525 nm.

3.Analysis of Obtained Fluorescence Intensity Data to Evaluate WSP-5 Sensitivity to H₂S ^[2] (Reference Only)

(1) Experimental Preparation: Use black, clear-bottom 96-well plates for the experiment. Prepare a final volume of 200 μL in each well.

Prepare PBS containing 1 mM CTAB as the buffer for all experiments.

(2) Probe Solubilization: Dissolve the WSP-5 probe in PBS containing 1 mM CTAB to a final concentration of 10 μM.

(3) H₂S Donor Addition: Add increasing concentrations of Na₂S or DAT as the H₂S donor to the wells containing the WSP-5 probe. Concentrations range from 0 to 10,000 μM.

(4) Fluorescence Measurement: Measure the fluorescence intensity at regular intervals (e.g., every 30 seconds) using a SpectraMax M5 microplate reader. λex/em = 502/525 nm.

(5) Data Collection: Record the fluorescence intensity measurements over a period of 30 minutes.

Precautions:This experimental protocol provides guidance for detecting cellular H₂S using WSP-5 and subsequent fluorescence measurements. Adjustments can be made according to specific experimental requirements.

References:

[1] He T , Qin X , Jiang C ,et al.Tumor pH-responsive metastable-phase manganese sulfide nanotheranostics for traceable hydrogen sulfide gas therapy primed chemodynamic therapy[J].Theranostics, 2020, 10(6):2453-2462.

[2] Yingzhu Zhou, Dr. Federico Mazur,et al. Sensitivity and Selectivity Analysis of Fluorescent Probes for Hydrogen Sulfide Detection[J]. Chemistry – An Asian Journal
March 1, 2022.