I am trying to label the lipid bilayer with lissamine rhodamine B and encapsulate 5(6) carboxyfluorescein inside the liposome. can anyone let me know the concentration of both the dye and lipid concentration need to be used to get good confocal image of liposome vesicle proper red bilayer and green dye inside the vesicle.
For labeling bilayer with rhodamine PE, or any fluorescent lipid for that matter, you will want to use a very small amount of the probe, less than 1 mol%, so that its fluorescence will not be strong so as to mask the changes you want to observe.
Hi Mohana,
for lipid-bilayer labeled rhodamine, you need ~1-2% fluorescent lipid with respect to your liposomal composition and this will give you good confocal imaging. I used DOPE-labeled rhodamine for one of my studies. See link below.
For encapsulated CF, it may require some trial and error and will eventually depend on how much encapsulation efficiency you can get. For example, it may be prudent to start with ~2:1 or 1:1 CF:lipids (wt:wt) and then check how much is encapsulated.
http://www.sciencedirect.com/science/article/pii/S0168365915002333
Thanks Rafael and Rahul for your answers. I have used 0.5 mole% rhodamine PE and 5 micronolar CF but i am not getting the proper sharp boundary of red bilayer. It looks like compleate red ball. What is the proportion of dye and lipid.
Hi Mohana,
Usually 0.1% molar fraction of rhodamine PE should work fine (PE:Lipids at 1:10000).
You might have some variations in the efficiency depending on the type of lipids that you use, but you can start with that and if you couldn't get a proper image you can change the ratios.
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