ASSI-Leu

Operating Instructions (The following is a recommended experimental protocol for guidance only and should be adjusted according to your specific requirements)

1. Stock Solution Preparation
1.1 Solvent: 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: Serum-free medium or PBS is usually used.
2.2 Working Concentration: Reference range is 1-10 μM.
2.3 Notes: Adjust the working solution concentration as needed; prepare and use it immediately.

3. Staining Procedure
3.1 Sample Type: HepG2 cells
3.2 Incubation Conditions: Incubate at 37°C. An incubation time of 30-60 min is recommended to obtain a stable ratio signal. Avoid light during incubation.
3.3 Washing Step: After incubation, wash the cells 2-3 times with pre-warmed PBS or serum-free medium to remove free probes and reduce background fluorescence.

4. Control Setup
4.1 Unstained Control: Used to adjust instrument voltage and eliminate autofluorescence of cells.
4.2 Positive Control: Pre-treat cells with the LAP inhibitor Bestatin (HY-B0134) at 100 μM for 1 h, then add ASSI-Leu and incubate for 30 min.

5. Detection and Analysis
5.1 Instrument Type: Confocal microscope, fluorescence microscope.
5.2 Wavelengths: Excitation wavelength is 405 nm; Channel 1 collects fluorescence at 410-470 nm (blue, enol-form emission), and Channel 2 collects fluorescence at 490-560 nm (yellow, keto-form emission).
5.3 Result Analysis: After ASSI-Leu enters cells, with the extension of incubation time, the blue fluorescence in Channel 1 gradually weakens while the yellow fluorescence in Channel 2 gradually increases, and the fluorescence ratio (Y/B) rises from approximately 0.79 (at 10 min) to approximately 2.47 (at 60 min). The yellow fluorescence is mainly localized in lysosomes (co-localized with LysoTracker Red (HY-D1300), with a Pearson correlation coefficient of 0.67).

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