I try to use inverted emulsion method to make vesicles. I know there are a lot of pubulications use this method, so I choose one of those to make vesicles. I use DOPC and mineral oil to make lipid mixtureI(sonication2h). 0.35mg/mL for emulsion and 0.8mg/mL DOPC for interface. Sucrose and glucose to make internal and exterior solution. Centrifuge force I used from RCF 0.2k-5k, 10-30min. However, I get vesicles sometimes , but not so many. And normally no vesicles at all, only miclles. Does anyone know which procedure is crucial step that I made in wrong way? Or from your experience, how to increase success rate to obtain more vesicles. Thanks a lot !
Update: I increase the concentration of lipid to 2mg/mL still not working. I red some literature use even lower one but still works fine. By the way, I use vortex method to make emulsion.
My first impression would be that your oil content is too high.
If I understand you correctly then you use roughly (based on molecular weight) an equal amount of oil (faty acids etc.) and phospholipid.
Only the phospholipid will form a bilayer while your oil components will not.
So my suggestion would be to lower the oil content and see whether you get better results.
Good luck!
Thanks a lot for sharing your experience Rob Keller . I will try to increase the lipid content!
Update: I increase the concentration of lipid to 2mg/mL still not working. I read some literature use even lower one but still works fine. By the way, I use vortex method to make emulsion. Rob Keller Entedhar Sarhat
I'm a bit puzzled, I wonder how you conclude that you only find micelles.
At least for the phospholipid part it will be virtually impossible to form micelles (did you try using only phospholipid).
If you are sure about your findings then I can only come up with one thing.
Possibly it has something to do with the sucrose and glucose you use.
Although it is thought that carbohydrates stabilize membrane structures. See for example:
https://www.sciencedirect.com/science/article/pii/0005273684900178
It is however also known that trehalose seems to destabilize bilayers:
https://www.sciencedirect.com/science/article/pii/0009308494900817
PS. As mentioned in the paper introduction of some Mg2+ seems to counteract such possible effects.
Hope this is of any help. Please let us know when you solved this mystery.
Yes, really confused. I check them under fluorescent microscope, either micelles or small vesicle. Actually should be small vesicle( due to the resolution of micrscope) Because I am looking for giant vesicle, that it not useful for me.
At first I thought probably because of milli Q water has unstable pH, then I start using PBS buffer. But still not working.
Thanks, I will read the literature. Rob Keller
PS. I just saw that you use in the beginning of your procedure sonication. You could try to skip this and see whether it improves the size of your vesicles.
Good luck with your work.
The sonication is for making the lipid mixture instead of making emulsion. I use vortex method to make emulsion.
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