Hello Fan Janice

When choosing a cell membrane dye for live-cell imaging alongside Hoechst nuclear staining you will have to consider the following.

1. The dye should not be toxic and should not interfere with cellular function.

2. The dye should provide clear and distinct visualization of the plasma membrane.

3. The dye should be stable under prolonged exposure to excitation light during live-cell imaging. It should be retained on the cell surface, and not rapidly internalized.

4. The dye should be spectrally distinct from Hoechst to allow for simultaneous imaging of both the nucleus and the membrane.

5. The dye should remain fluorescent and stable over the duration of the experiment.

Keeping these criteria in mind, I would recommend the use of MemBright dyes (like BioTracker MemBright 560, BioTracker MemBright 640, and BioTracker MemBright 488) or lipophilic carbocyanine dyes (like DiI (DiIC18(3)) and DiO (DiOC18(3)) as they are highly photostable, lack cytotoxicity, and are able to clearly label the plasma membrane. These dyes offer good membrane detail and are compatible with long-term monitoring, allowing for stable fluorescence over time. Both MemBright and lipophilic carbocyanine dyes can be used in co-culture experiments to visualize the membranes of different cell types.

For co-culture experiments requiring long-term live imaging and minimal toxicity, MemBright dyes are generally preferred due to their better retention and lower toxicity compared to many conventional lipophilic carbocyanine dyes. The choice among different MemBright dyes depends on the desired emission wavelength and your microscope's filter sets. MemBright dyes offer a range of colors, from orange to near-infrared. Some popular options include:

MemBright 488: Emitting in the green spectrum, suitable for excitation at 488 nm and emission range of 500-550 nm.

MemBright 560: Emits red/orange fluorescence with excitation at 560 nm and emission range of 567-750 nm.

MemBright 640: Emits in the far-red spectrum, ideal for excitation at 640 nm and emission range of 640-800 nm.

You may think of including MemBright dyes in the list, in addition to PKH 26 and PKH 67, and choose the best that works for your experiment. I have not worked with PKH26 and PKH67 and would not be in a position to comment.

Best,

Fan Janice

PKH26 and PKH67 are recommended. However, PKH67 is more stable, while PKH26 powder and solution are unstable and need to be stored in nitrogen.

The answer to this question comes from MedChemExpress (MCE) Technical Support.

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Malcolm Nobre Thank you so much for your advice, I will definitely consider using dyes from the Membright family along with the two other I mentioned earlier.