Preparation and imaging of lipid bilayer-coated silica microspheres

Prepare Tris buffer: 100 mM NaCl, 10 mM Tris base, pH 7.4

Prepare Tris-Ca2+ buffer: 100 mM NaCl, 3 mM CaCl2, 10 mM Tris base, pH 7.4

Filter 50 ml of Tris buffer and Tris-Ca2+ buffer using a 0.02 μm syringe filter (6809-1102, Whatman).

Dissolve DPPC (850355C, Avanti Polar Lipids) in chloroform (366927, Sigma-Aldrich) to a concentration of 25mg/mL in a glass vial with a PTFE (Teflon) lined cap (14-955-327, Fisher Scientific). You will need 23µL per sample.

Dissolve cholesterol (C8667-5G, Sigma-Aldrich) in chloroform (366927, Sigma-Aldrich) to a concentration of 10mg/mL in a glass vial with a PTFE (Teflon) lined cap (14-955-327, Fisher Scientific). You will need 20µL per sample.

Prepare a DPPC + 40% cholesterol mixture by combining 23µL 25mg/mL DPPC with 20µL 10mg/mL cholesterol in a new glass vial with a PTFE (Teflon) lined cap (14-955-327, Fisher Scientific).

Evaporate the solvent under vacuum.

Re-hydrate the lipids/cholesterol mixture using 1mL of Tris-Ca2+ buffer (100millimolar (mM) NaCl, 3millimolar (mM) CaCl2, 10millimolar (mM) Tris base, 7.4).

0h 40m 0s Vortex for 0h 0m 30s.

Sonicate the solution using a tip sonicator for 0h 40m 0s (cycles of 45 seconds on and 15 seconds off, 60% amplitude) until the solution runs clear.

Centrifuge the solution for 0h 1m 30s at 14000rcf,0h 0m 0s to remove residu from the sonicator probe. Keep the supernatant.

Dilute the glass beads (5 µm diameter, 44054-5ML-F, Sigma-Aldrich) to approximately 2.8mg/mL.

Clean the beads by centrifuging and replacing the supernatant with Tris-Ca2+ buffer.

Heat the glass beads and the lipid vesicle solutions to 65°C using a heated water bath.

Once heated to 65°C, mix the glass bead and lipid vesicle solution together in a 1:1 ratio. Leave the mixture at 65°C and wait for 0h 30m 0s.

Turn the heating element of the water bath off, and let the glass bead/lipid vesicle mixture slowly cool down to room temperature inside the water bath. The lipid vesicles will attach to the glass beads, open and form a lipid-bilayer on the glass surface of the beads.

Gradually replace the buffer by Tris (100 mM NaCl, 10 mM Tris base, pH 7.4):

Centrifuge for 0h 5m 0s at 0.3rcf,0h 0m 0s.

Gently replace two thirds of the supernatant with Tris.

Repeat the last two steps (14.1 and 14.2) a total of 6 times.

Store the lipid-coated glass beads at 4°C and use as soon as possible, or within a week of preparation.

Argon plasma clean cover glass (VWR collection, 631-0124) for 0h 30m 0s in a plasma cleaner (Expanded Plasma Cleaner, PDC-002, Harrick Plasma).

Create a sample well on the cleaned cover glass by sticking a frame-seal slide chamber (9x9 mm, SLF0201, Bio-rad) on the cover glass.

Pipet 70µL of 0.01% PLL (0.01% poly-L-lysine solution, P4707, Sigma-Aldrich) into the well and wait for 0h 15m 0s. The PLL will coat the surface of the cover glass.

Use a pipet to remove the excess PLL from the well and immediately replace it with 70µL of filtered PBS.

Use a pipet to remove the excess filtered PBS from the well and immediately replace with 70µL filtered PBS. Gently pipet up and down in the corners of the well. Repeat this step 2 more times.

Use a pipet to remove the excess PBS from the well and immediately replace with 50µL of the lipid bilayer-coated beads. Wait for 0h 5m 0s.

Use a pipet to gently remove the excess PBS from the well and immediately replace with 50µL of a dye of choice, e.g. 1nanomolar (nM) Nile red for PAINT of the lipid membrane.

Image the sample the same day.