Mem-pH

Guidelines (The recommended experimental protocol below is for guidance only and should be adjusted according to your specific requirements)

1. Stock Solution Preparation
1.1 Solvent: DMSO.
1.2 Recommended concentration: 1 mM.

2. Working Solution Preparation
2.1 Diluent: Opti-MEM.
2.2 Working concentration: 1 µM.
2.3 Notes: Adjust the working solution concentration as needed; prepare and use immediately.

3. Staining Procedure
3.1 Sample type description: Adherent cells (KB cells, human epithelial cancer cells)^[1][2]
3.1.1 Adherent cells: No trypsin digestion is required before staining.
3.2 Incubation conditions: Incubate cells with 1 µM Mem-pH at 37°C for 1 h.
3.3 Washing step: Wash cells once with Opti-MEM after incubation.

4. Control Setup
4.1 Plasma membrane colocalization control: After staining with Mem-pH and washing, add 200 nM MemBright-640 and image immediately.
4.2 Lysosome colocalization control: After staining with Mem-pH and washing, incubate cells in Opti-MEM at 37°C for 24 h, followed by staining with 1 µM Cresyl Violet, washing with Opti-MEM, and then imaging.

5. Detection and Analysis
5.1 Instrument type: Laser scanning confocal microscope.
5.2 Excitation/emission wavelengths:
5.2.1 Alkaline form (green channel): Excitation wavelength 405 nm, emission wavelength 450-550 nm.
5.2.2 Acidic form (red channel): Excitation wavelength 488 nm, emission wavelength 500-700 nm.
5.3 Result analysis:
5.3.1 Changes in fluorescence intensity: During vesicle acidification, the green fluorescence intensity decreases while the red fluorescence intensity increases; the alkaline form shows high green fluorescence brightness at the plasma membrane and early endosomes.
5.3.2 Fluorescence localization: Initial fluorescence localizes to the plasma membrane; over time, fluorescence accumulates in intracellular vesicles (early endosomes, late endosomes, lysosomes) via endocytosis.
5.3.3 Color changes: Green fluorescence corresponds to neutral/alkaline pH (≈7.0, plasma membrane/recycling endosomes); yellow fluorescence corresponds to intermediate pH (≈5.0, early endosomes); red fluorescence corresponds to acidic pH (≤4.6, lysosomes).
5.3.4 Ratio analysis: Quantify vesicle pH by calculating the ratio of red/green fluorescence intensity.

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