Silica microspheres

Pulldowns were performed as previously described (46 (link)). Briefly, silica microspheres (Bangs Laboratories) were coated with a bilayer composed of either phosphatidylcholine (PC) and phosphatidylserine (PS), at a molar ratio of 80:20 PC:PS, or phosphatidylcholine (PC), phosphatidylinositol (PI) and phosphatidylinositol 4,5-bisphosphate (PIP2), at a 48:48:4 molar ratio of PC:PI:PIP2. All lipids were purchased from Avanti Polar Lipids. Indicated proteins were anchored to these bilayers, prior to incubation in cell-free porcine brain extract. Following incubation for 15 min, bilayers were washed extensively and associated proteins were analyzed by SDS-PAGE. To identify recruited proteins, beads were washed with 50 mM ammonium bicarbonate (pH 8.5) supplemented with 0.5 mM dithiothreitol (DTT) and then were incubated with trypsin. The digested proteins were identified by electrospray ionization liquid chromatography mass spectrometry (MS) (Mass Spectrometry Service, Cambridge Centre for Proteomics, University of Cambridge, Cambridge, UK). The MS fragmentation data were used to search the National Center for Biotechnology Information (NCBI) database using the MASCOT search engine (www.matrixscience.com).

L4 hermaphrodites were picked to a fresh plate and upshifted to 25°C for approximately 24 hr prior to each FRAP experiment. Worms were placed on slides with 7% agarose pads, and a mixture of 1 μl of silica microspheres (Bangs Laboratories, Cat# SS02N/11167) and 0.4–0.6 μl of anesthetic solution (0.2% tricaine + 0.02% tetramisole) was added to the pad. For experiments done on the Deltavision OMX microscope (see below), 0.5 μl of serotonin solution [57 ] (final concentration of 25mM prepared from Serotonin Hydrochloride salt, Sigma) was added together with the silica beads and anesthetic solution. 5–7 worms were then mounted with a small amount of bacteria (in order to maintain healthy conditions during imaging). The coverslip (22X40mm-1.5) was sealed to the slide using a thin line of petroleum jelly applied by syringe around the agarose pad; mild pressure was applied to achieve an even spread of liquid on the pad and to ensure efficient immobilization of the worms. After imaging, the cover slip was removed and worms were transferred to a seeded NGM plate and allowed to recover for 1–2 days; worms were monitored for movement and egg laying to verify their recovery.

Nanoparticle tracking analysis (NTA) was performed using a NanoSight300 (NS300, Malvern Instruments ltd, Malvern, UK). The naEVs fraction isolated from E4.5 spent media was resuspended in 1 mL of sterile PBS w/o Ca2+/Mg2+ (Biowest, Nuaillé, France, ref. L0615-500). In parallel, an aliquot of fresh embryo culture media was processed as a blank control. Three 60 sec videos were recorded under the static flow conditions for each sample with camera level set at 11. Videos were analyzed with NTA software version 3.2 Dev Build 3.2.16 to determine mean size and estimated concentration of measured particles with corresponding standard error. The NS300 system was calibrated with silica microspheres 100, 167, and 300 nm (Bangs Laboratories, Inc.; Fishers, IN, USA) prior to analysis, as previously demonstrated [30 (link)], auto settings were used for blur, minimum track length, and minimum expected particle size.

Polymer and lipid reagents were purchased from commercial venders and stored and used according to the recommendations of the vendor: 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC; Avanti Polar Lipids); poly(ethylene oxide-b-butadiene) (EO₂₂Bd₃₃; P40494B, Polymer Source). Deionized (DI) water was prepared using a Millipore Synergy UV-R water purification system. Tris buffer solutions (10 mM tris(hydroxymethyl)aminomethane and 100 mM NaCl) were prepared and titrated to their appropriate pH using HCl or NaOH, while controlling for osmolality (200 +/−10 mOsm). Silica microspheres (0.163 micrometers, Bangs Laboratories) were diluted to 1 mg mL⁻¹ in Tris buffer solutions at pH values ranging from 2 to 12.

The Silica microspheres employed in this experiment was purchased from Bangs Laboratories, Inc. (Fishers, IN, USA). The Sio2 microsphere (beads) were available as an aqueous suspension. The diameter of individual beads was 0.5 µm with a density of 2 g/cm³. Due to the high concentration of beads, they were diluted 10⁴ times by serial suspension in purified water. We conducted parallel experiments under the same experimental conditions. Therefore, no attempt was made to wash the beads and separate them from their liquid phase prior to the serial dilution of the beads. We refer to these beads diluted by water as “prepared beads”.