TPE-Cy

Guide (The following is our recommended protocol. This protocol is for guidance only and should be modified according to your specific needs).

1. Stock Solution Preparation
1.1 Solvent: Chloroform, tetrahydrofuran, methanol, ethanol, DMSO.
1.2 Concentration recommendation: 10 μM, 50 μM.

2. Working Solution Preparation
2.1 Diluent: Cell culture medium.
2.2 Working concentration: 10 μM.
2.3 Note: Adjust working solution concentration as needed; prepare fresh before use.

3. Staining Procedure
3.1 For aqueous buffer solution samples[1]:
3.1.1 Incubation conditions: Incubate with TPE-Cy at 10 μM or 50 μM concentration.
3.2 For adherent cells[2]:
3.2.1 Incubation conditions: Incubate cells with 10 μM TPE-Cy working solution for 2 h.
3.2.2 Washing steps: Wash cells to remove unbound dye after incubation.

4. Controls
4.1 Set up unstained cell control for flow cytometry analysis.

5. Detection & Analysis
5.1 Instrument type: Fluorescence spectrometer, confocal fluorescence microscope, flow cytometer.
5.2 Ex/Em wavelengths:
5.2.1 Fluorescence spectrometer: Ex = 380 nm, Em = 630 nm (red emission); Ex = 380 nm, Em = 480 nm (blue emission); Ex = 440 nm, Em = 625 nm.
5.2.2 Confocal fluorescence microscope: Blue channel - Excitation at 405 nm; Red channel - Excitation at 488 nm, emission collected at 565−671 nm.
5.2.3 Flow cytometer: Blue channel - Excitation at 375 nm, emission at 450±20 nm; Red channel - Excitation at 488 nm, emission at 610±11.5 nm.
5.3 Result analysis:
5.3.1 Fluorescence intensity changes: Strong red emission at pH < 5; strong to moderate red emission with decreasing intensity at pH 5-7; weak to no red emission at pH 7-10; no emission to strong blue emission with increasing intensity at pH 10-14; strong blue emission at pH > 14; emission intensity shows linear relationship with pH in 5-7 range; blue emission intensity increases with higher pH; red emission intensity increases with lower pH.
5.3.2 Color changes: Red fluorescence at acidic/neutral pH; blue fluorescence at basic/alkalescent pH.
5.3.3 Fluorescence localization: Red emission localizes in acidic organelles (lysosomes); blue emission localizes in alkalescent organelles (mitochondria) and cytoplasm.
5.3.4 Ratiometric analysis: Generate pseudo-color ratiometric images where red corresponds to low blue-to-red ratio (acidic) and blue corresponds to high blue-to-red ratio (alkalescent); the ratio of blue-to-red emission intensity correlates with local pH, decreasing in acidic conditions and increasing in alkalized conditions.
5.3.5 Flow cytometry analysis: Differentiate stained cells from unstained controls; use blue-to-red emission ratio to assess intracellular pH shifts.