Lipid Coated Bead Preparation
From Heit Lab Wiki
- In a glass container, and using lipids dissolved in chloroform, mix desired lipids to a final quantity of ~4mmol total lipid.
- Dry lipids under inert gas.
- Suspend lipids in 400ml PBS or other buffer, sonicate to suspend completely.
- Note: if covered with inert gas, these lipids are stable for ~1 week at 4oC.
Glass Bead Preparation:
- Measure out 2mg of glass beads of the desired diameter.
- Wash 3X in 1ml ddH2O.
- Pellet glass beads. Remove as much water as possible.
- Suspend beads in 400ml of the PBS-lipid solution, rotate at room temp 20min to allow bylayers to self-assemble.
- Pellet beads with a brief (<5sec) cetrifugation and remove the liquid phase. This will remove unincorporated liposomes.
- Suspend beads in 200-400ml desired buffer or in ddH2O.
Laxex Bead Preparation:
- Measure out 10ml latex beads of the desired diameter.
- Wash 2X in 50mM carbonate-bicarbonate buffer, pH 9.6. Pellet cells with a max-speed spin in minifuge, 10min.
- Incubate 2hrs at 37oC, rotating constantly, with lipisome fractions prepared in “lipid preparation”.
- Wash 2X with carbonate-bicarbonate buffer
- Incubate with 5% BSA in PBS for 2+ hours to block beads.
- 1.15g Anhydrous sodium carbonate
- 4.13g Sodium bicarbonate
- 100ml ddH2O
Dissolve in water, pH to 9.6.