Prepare your liposomes by rehydration of a dry lipid film containing 1 mol% of DiO. No separation of unincorporated fluorescent lipid by centrifugation is necessary.

Reference:

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Dear Adam

Thanks for your answer. Do you know whats the reason that for some dyes such as methylene blue they do centrifugation to get rid of excess dye but there is no need to centrifuge for dyes such as 3 3'-dioctadecyloxacarbocyanine perchlorate (Dioc18)? I guess the mechanism in which the dye gets incorporated with liposomes is different for these two dyes. Is that right? If yes could you please explain the difference?

Thank you

Yes, the mechanism is different. DiO is essentially a lipid, whereas methylene blue is a small water-soluble molecule. Small water-soluble molecules can be incorporated into the aqueous interior compartment of liposomes, but not into the lipid bilayers. Lipid-like molecules like DiO are incorporated into the lipid bilayer.