TMA-DPH, a hydrophobic fluorescent membrane probe (Ex: 355 nm; Em: 430 nm), is used for studying membrane dynamics and structure.

The TMA-DPH fluorescence lifetime remains constant, 6.2±0.2 ns, below the same critical concentration of 2 μM, but decreases significantly with higher concentrations. This decrease, however, slows down above 5 μM. The TMA-DPH fluorescence anisotropy displays a similar evolution to the fluorescence lifetime. It is first constant, 0.283±0.003, again until 2 μM, and then decreases rapidly to 0.270±0.003 with 5 μM, continuing to fall, but at a lower rate with higher concentrations [2].

I. Determination of cell membrane fluidity by fluorescence polarization
1. Solution preparation: Dissolve TMA-DPH in an appropriate solvent (such as DMSO) to a concentration of 10-20 μM. The concentration can be optimized according to experimental needs.
2. Cell treatment: Add TMA-DPH solution to cultured cells, usually at a concentration of 0.1-2 μM. The treatment time is 15-30 minutes to ensure that the probe can be evenly embedded in the cell membrane.
3. Fluorescence measurement:
1) Use a fluorescence polarization (fluorescence polarization) device with an excitation wavelength set to 355 nm and an emission wavelength set to 430 nm to measure the fluorescence polarization value.
2) Record the fluorescence polarization values ​​at different time points or under different conditions to analyze the changes in cell membrane fluidity.
4. Data analysis:
Analyze the fluidity of the cell membrane based on the changes in fluorescence polarization values. A higher polarization value indicates that the cell membrane is more solid (lower fluidity), while a lower polarization value indicates that the membrane has higher fluidity.
2. Analysis of membrane region fluidity:
1. Selection of membrane regions: Specific labeling or sorting of different membrane regions (such as plasma membrane, endoplasmic reticulum membrane, etc.) can be performed. Single-layer membrane or double-layer membrane can be used as the research object.
2. Experimental design: TMA-DPH is added to the cell culture medium, and the fluorescence polarization of specific regions is detected by appropriate techniques (such as confocal microscopy, fluorescence microscopy).
3. Analysis of data: Comparing the fluorescence polarization values ​​of different membrane regions can study the differences in fluidity of different membrane regions.