Hi everyone,
I recently performed Laurdan staining on live cells using a 10 µM working solution diluted in complete culture medium using Laurden from MCE (HY-D0080). I incubated the cells at 37°C for 1 hour, protected from light, and then fixed them with 4% paraformaldehyde for 15 minutes before imaging them on a confocal microscope. I used 440 nm and 488 nm excitation wavelengths, expecting to observe membrane labeling with yellow edges due to Laurdan’s spectral shift. However, the fluorescence signal was very weak, and the expected membrane outlining effect wasn’t clearly visible.
I’m wondering:
- Is 1 hour of staining sufficient, or should I extend the incubation time?
- Could fixation with paraformaldehyde be affecting the Laurdan signal?
- Is there an optimal buffer or staining condition I should consider?
- Could the weak signal be due to Laurdan's sensitivity to membrane phase rather than just presence?
Any advice or insight would be greatly appreciated. Thank you!
Fan Janice Hi, I reviewed the relevant literature, where the typical experimental concentration is 10 μM, and the incubation time generally ranges from 10 to 30 minutes. Your treatment time seems to be longer than usual, so I would suggest adjusting it based on the references:
] https://pmc.ncbi.nlm.nih.gov/articles/PMC11803045/ (37°C, 10 μM, incubation for 10 min) [2] https://www.mdpi.com/2079-6382/13/9/882 (Room temperature, 10 μM, 20 min) [3] https://pmc.ncbi.nlm.nih.gov/articles/PMC11497084/ (37°C, 10 μM, 30 min)
Additionally, you mentioned that you want to observe yellow fluorescence at the cell edges to reflect membrane phase state, but the current results cannot be observed with the naked eye. In that case, you could use excitation wavelengths of 440 nm and 490 nm to indicate ordered membranes (lipid raft regions) and disordered membranes (non-lipid raft regions), respectively. You can then calculate the GP image based on the obtained results and validate the fluorescence ratio.
GP = [I(440 nm) - G · I(490 nm)] / [I(440 nm) + G · I(490 nm)]
The answer to this question comes from MedChemExpress (MCE) Technical Support. [email protected]
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