CSMPP

Guidelines (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: Dimethyl sulfoxide (DMSO).
1.2 Concentration Recommendation: It is generally recommended to prepare a high-concentration stock solution of 1-10 mM.

2. Working Solution Preparation
2.1 Diluent: Serum-free medium or PBS is usually used. Proteins and esterases in serum may interfere with staining results or cause dye hydrolysis.
2.2 Working Concentration: 2.5-10 μM.
2.3 Notes: Adjust the working solution concentration as needed; prepare and use the solution immediately.

3. Staining Procedures
3.1 Description of Sample Types
3.1.1 Adherent cells: HeLa cells, ARPE-19 cells, zebrafish (ZF4) cells.
3.1.2 Suspension cells (oyster hemocytes): Collect hemocytes and incubate with CSMPP working solution
3.2 Incubation Conditions
3.2.1 HeLa cells: Incubate with 5 μM CSMPP at 37 °C in 5% CO₂ for 15 min, then wash with PBS and allow internalization for 30 min.
3.2.2 Zebrafish ZF4 cells: Incubate with 2.5 μM CSMPP at 28 °C for 45 min, then wash with PBS 3 times.
3.2.3 Oyster hemocytes: Incubate with 2.5 μM CSMPP at 20 °C in the dark for 1 h.
3.3 Washing Procedures
3.3.1 HeLa cells: Wash once with PBS and allow internalization for 30 min.
3.3.2 ZF4 cells: Wash with PBS 3 times.
3.3.3 Oyster hemocytes: Wash with PBS 2 times.

4. Control Setup
4.1 Unstained control: Used to adjust instrument voltage and eliminate autofluorescence of cells.
4.2 Positive control: Bafilomycin A1 (HY-100558): Treat cells with 50 nM Bafilomycin A1 for 30 min.
4.3 Blank control: Replace the dye working solution with an equal volume of solvent to eliminate fluorescence interference from the reagent itself.

5. Detection and Analysis
5.1 Instrument Types: Laser scanning confocal microscope, fluorescence spectrophotometer, UV-visible spectrophotometer.
5.2 Wavelengths: For confocal microscopy, excitation at 405 nm, green channel at 470-560 nm (or 400-555/416-555 nm), red channel at 560-700 nm (or 557-704 nm); for spectroscopy, excitation at 365 nm, emission peaks at 503/615 nm.
5.3 Result Analysis
5.3.1 Changes in fluorescence intensity: Aggregation state enhances fluorescence by 7-fold (AIE property); emission redshifts from 503 nm to 615 nm (112 nm redshift) with decreasing pH; the process is reversible over 5 cycles of pH 7↔3.
5.3.2 Fluorescence localization: Targets lysosomes; the normal lysosomal pH of HeLa cells is 5.06; cell viability remains >80% at concentrations ≤10 μM.
5.3.3 Color changes: Green fluorescence (503 nm) under neutral/basic conditions, red fluorescence (615 nm) under acidic conditions; an increased red/green ratio indicates lysosomal acidification.