Hi fellow researchers,
I am using bodipy green to stain lipid in my cells. But I recently acquired a cell line that has EGFP construct stably integrated, so it is very important that I have a red lipid stain. In the lab I have Bodipy red (558/568 C12), and Nile red, but both of them bleed through strongly into a green channel. Concentrations I used for them range from 1/1000 to 1/50,000 (from 1nM to 0.5nM). No matter what concentration I use it bleeds through! Stock (1mM) was prepared in ethanol just few days after they were ordered, so I assume my dye is okay. I just need to find the concentration or condition it works!
Does anyone have any experience using these dyes for lipids? Please share your tricks and help a poor grad student. Thank you!
Hi, I think it is not a problem with your dyes. For trouble shooting the optical path should be checked, maybe your emission filters are not stringent enough. To solve the problem fast image prossessing via spectral unmixing ( e.g. ImageJ) would be the easyest way in my eyes.
Regards
http://rsb.info.nih.gov/ij/plugins/spectral-unmixing.html
I agree with Benjamin that you probably need to check your filters and dichroics (if relevant). Most companies, like Semrock, allow you to download the data for their filters, so you can estimate the bleed-through. If your two fluorophores are very different in quantum yield of emission or absorption cross-section at your excitation wavelength, then you need plan more carefully. Even 1% bleed-thru can drown out the signal from a very weak (or not well-excited) emitter.
Thanks for your input. I always have separate channels and did confocal analysis for all my samples and still in some samples there was bleed through. I tried lowering the working concentration and instead go higher with laser and gain master. This reduced bleed through. Thank you again.
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