LRhod-PM

Operating Instructions
(The following is a recommended experimental protocol for reference only, and adjustments are required based on specific needs)

1. Stock Solution Preparation
1.1 Solvent: Methanol.
1.2 Recommended concentration: 10 μM.

2. Working Solution Preparation
2.1 Diluent: PBS.
2.2 Working concentrations: 200 nM (for confocal imaging); 20 nM (for live single-molecule localization microscopy).
2.3 Notes: Adjust the working solution concentration as needed; prepare fresh immediately before use.

3. Staining Procedure
3.1 Sample types: Adherent cells (HeLa, A549); suspension/differentiated cells (THP-1 macrophages)^[1].
3.1.1 Adherent cells: No trypsin digestion is required for wash-free staining.
3.2 Incubation conditions:
3.2.1 For confocal imaging: Incubate cells with 200 nM LRhod-PM for 5 min.
3.2.2 For photoactivation: Irradiate with a 552 nm laser (10% power) for no more than 40 s.
3.2.3 For live single-molecule localization microscopy imaging: Irradiate with a 532 nm laser (0.25 kW/cm²).
3.3 Washing step: No washing required (wash-free staining).

4. Control Experiments
4.1 Light-free control: Perform the oxidation experiment of LRhod-DA under light-free conditions to confirm its light-dependent activation property.
4.2 Negative control: Use unirradiated LRhod-PM to verify its non-fluorescent off-state.
4.3 Positive control: Use Rhod-PM to verify cell membrane localization effect and fluorescent signal.
4.4 Colocalization control: Co-stain with MemBright probes (MemBright-Cy5.5 (HY-D0924), MemBright 488) to confirm its cell membrane targeting property.

5. Detection and Analysis
5.1 Instruments:
5.1.1 Laser scanning confocal microscope: Excitation light (Ex) 552 nm, emission light (Em) 585 nm (The maximum excitation/emission wavelengths of Rhod-PM are 560/585 nm).
5.1.2 Single-molecule localization microscope: Excitation light (Ex) 532 nm, emission light (Em) 585 nm (The maximum excitation/emission wavelengths of Rhod-PM are 560/585 nm).
5.2 Result analysis:
5.2.2 Fluorescence intensity change: The fluorescence intensity can increase by up to 5-fold after irradiation and reaches a plateau within approximately 30 s; continuous blinking events maintain stable localization counts.
5.2.2 Fluorescence localization: Fluorescence is restricted exclusively to the cell membrane, including fine protrusive structures such as filopodia and tunneling nanotubes.
5.2.3 Color change: After photoactivated oxidation into Rhod-PM, LRhod-PM emits pink-purple fluorescence.
5.2.4 Super-resolution performance: The resolution of live single-molecule localization microscopy imaging reaches ~77 nm (in comparison, the resolution of wide-field imaging is approximately ~681 nm), with an average localization precision of 21.1-37.9 nm.

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