pH-Ap-1

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: DMSO.
1.2 Concentration recommendation: 10 mM.
2. Working Solution Preparation
2.1 Diluent: Cell culture medium.
2.2 Working concentration: 2.5 μM.
2.3 Note: Adjust working solution concentration as needed; prepare fresh before use.
3. Staining Procedure
3.1 Sample type: Adherent cells (SKOV-3 cells).
3.1.1 Adherent cells: Trypsinization is not mentioned for staining steps.
3.2 Incubation conditions: Incubate cells with 2.5 μM pH-Ap-1 working solution for 30 min.
3.3 Washing steps: Replace the medium with high K+ buffer solutions containing igericin (HY-127019) and Monensin (HY-N4302) for intracellular pH calibration.
4. Controls
4.1 Set up normal (untreated) SKOV-3 cells as a control.
5. Detection & Analysis
5.1 Instrument: Confocal fluorescence microscope.
5.1.1 Ex = 488 nm; Em = 500–550 nm (green channel), 625–750 nm (red channel).
5.2 Result analysis:
5.2.1 Fluorescence intensity changes: In acidic conditions (lower pH), red fluorescence intensity increases while green fluorescence intensity decreases; in alkaline conditions (higher pH), green fluorescence intensity increases while red fluorescence intensity decreases. The ratio (I_Green/I_Red) shows a linear increment in the intracellular pH range of 5.46 to 7.46.
5.2.2 Fluorescence localization: Red fluorescence (acidic regions) appears as spots in cells, with larger spots observed in autophagy-induced cells.
5.2.3 Color changes: Red fluorescence at acidic pH, green fluorescence at alkaline pH, with yellow-green fluorescence in slightly acidified cells.
5.2.4 Quantify intracellular pH using the pre-established calibration curve (I_Green/I_Red vs. pH) to measure pH fluctuations during autophagy.