TMA-DPH (Synonyms: N,N,N-Trimethyl-4-(6-phenyl-1,3,5-hexatrien-1-yl)phenylammonium (p-toluenesulfonate))

This protocol is only a guide. Please modify it according to your specific needs.

1. Preparation of staining solution

It is recommended to use DMSO to dissolve the powder at a concentration of 1~5mM, and then use a suitable buffer (such as serum-free culture medium, HBSS or PBS) to dilute the DMSO solution to prepare a dye working solution with a concentration of 0.5~5μM.

Note:

① This product is unstable in solution. It is recommended to prepare it immediately before use.

② Please adjust the concentration of the working solution according to actual conditions.

2. Cell suspension staining

(1) Suspended cells: Collect cells by centrifugation at 4°C, 1000-1500rpm, and wash twice with PBS for 5 minutes each time.

(2) Adherent cells: Discard the culture medium and add trypsin to digest the cells. Discard the supernatant by centrifugation at 4°C, 1000-1500rpm, and wash twice with PBS for 5 minutes each time.

(3) Resuspend the cells in TMA-DPH working solution (e.g., 2 μM), incubate for a short period of time (e.g., 10 seconds) at room temperature in the dark, and then immediately transfer the unwashed cells to a plate and observe under a microscope.

3. Cell adhesion staining

(1) Discard the culture medium and wash twice with PBS for 5 minutes each time.

(2) Cytoplasmic membrane staining: Incubate the cells with TMA-DPH working solution at a final concentration of 2 μM at room temperature for a short period of time (10 seconds), and then transfer the unwashed slide to a microscope for observation.

(3) Cell internalized membrane marker staining: Transfer the cells to a slide flask and incubate with TMA-DPH at a final concentration of 2 μM at 37°C for the required time according to the experimental requirements. After incubation, gently shake the slide in PBS for a few seconds to wash to eliminate peripheral markers.

Note: The optimal incubation time varies for different experiments. Please modify it according to the experimental requirements.

4. Fluorescence detection: The excitation/emission light of TMA-DPH is 355/430nm respectively.

Notes:

① TMA-DPH will immediately integrate into the plasma membrane after contacting the cell, and then accumulate in lysosomes and highly acidic late endosomes.

② The fluorescence lifetime of TMA-DPH remains constant at 6.2±0.2 ns when the concentration is lower than 2μM; when the concentration is higher than 2μM, it will decrease significantly with the increase of concentration; when it exceeds 5μM, the rate of decrease decreases.

③ Fluorescent dyes all have quenching problems, please try to avoid light to slow down fluorescence quenching.

④ For your safety and health, please wear a lab coat and disposable gloves when operating.

References:

[1]. Illinger, D., and Kurhy, J.G. The kinetic aspects of intracellular fluorescence labeling with TMA-DPH support the maturation model for endocytosis in L929 cells. Journal of Cell Biology 125(4), 783-794 (1994).