Hi all,
I have tried to do a calcein leakage assay by LUVs that I made (dopc:dopg 1:3, dopc:dopg 3:1, and dopc only). In doing so I dissolved my lipids film in 2 mL of 10mM tris buffer containing 70mM calcein and after incubation at RT for 1h and freez-thaw for 5 cycles, I extrude them by a mini extruder with 10mm filter support and 0.1um PC membrane. However, after centrifugation I got no pellet. But when I skipped the extrusion step and directly centrifuged my samples I got pellet. Thus, I believe my lipids are missed during the extrusion but I have no idea how should I overcome this issue. Does anyone have any idea?
Cheers,
Maryam
Hi Maryam,
It happens that a portion of your lipids gets stuck in your extruder. It also happens that some volume of the whole solution stays in the extruder, especially when the extruder is dry before usage.
Before extrusion, I always extrude an empty buffer a few times to fill in all the small voids in the extruder with the buffer and not to lose the sample. With this step, I also check if everything is tight and I do not have any spill.
Also, make sure that your extruder is properly cleaned before you use it. There might be some leftover molecules from the previous usage, which stick to your lipids and make it impossible to go through. TO properly clean it, use methanol multiple times, then use chloroform for the glass and metal parts, and then put everything to the sonication bath. You can sonicate it first in methanol, second in ethanol, and third in water right before you use it again to get rid of any organic solvents left in there. If there are organic solvents in the extruder, they will dissolve your lipids.
That's everything that comes into my mind. Good luck in making it work!
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