Preparation and characterization of Lumin-EX beads
Antibodies were conjugated to magnetic microspheres in selected color range, functionalized with carboxyl groups (MagPlexR, Luminex Corp., Cat. No. MC1XXXX-01), using 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) chemistry (He et al. 2007). Following conjugation, the resultant bead concentration (recovery) was determined in a Countess™ 3 FL automated cell counter (Thermo Fisher Scientific, Cat. No. A49866) using reusable chamber slides (Thermo Fisher Scientific, Cat. No. A25750). The beads were stored in a NeuroDex blocking buffer, which minimizes non-specific interactions, at a minimum concentration of 1x106/ml and for up to 3 months. To measure antibody loading, Lumin-EX beads were resuspended in Assay Diluent at 5.0104 beads/ml and loaded into 96-well black plates (BrandTech, Cat. No. 781671), a minimum of 200 beads/well. Serially diluted PE-conjugated secondary antibodies were added in duplicate. Assay Diluent only was used as a background control. The staining was carried out for 30 min at room temperature in a Genie® Microplate Mixer (600 RPM). Beads were washed three times in a magnetic plate holder and resuspended in xMAP Sheath Fluid Plus (100 µl/well, Thermo Fisher Scientific, Cat. No. 4050021). The plates were analyzed in a Luminex 200 plate reader, a minimum of 100 beads per condition.
Preparation of biotinylated detection antibodies
Antibodies were biotinylated by overnight incubation at -4oC with an EZ-link™ reagent (Thermo Fisher Scientific, Cat. No. 21442), at 20-fold molar excess, per manufacturer’s instructions. Excess reagent was removed using Zeba™ Spin Desalting Columns, 7K MWCO (Thermo Fisher Scientific, Cat. No. 89882).
sample where diluted (marked for each experiment) into a final volume of 50 µl in Assay Diluent and added into 96-well black plates. Assay Diluent only was used as a background control. Working suspension of Lumin-EX beads was generated to yield at least 2.5x104 beads/ml for each antibody/bead combination, for up to 7 IEL analytes, and 50 µl of working suspension was added to each well. EV capture was carried out overnight (16–18 hours) at 4oC on a microplate shaker (600 RPM). The plates were washed twice in a magnetic plate holder in NeuroDex Wash Buffer 1 and twice more in Wash Buffer 2, and the beads were resuspended in Assay Diluent supplemented with biotinylated detection antibody (1-2 µg/ml, 50 µl/well). After 2 hours incubation at room temperature, in a microplate shaker, the beads were washed 3 times in Wash Buffer 2, resuspended in 50 µl PBS supplemented with Streptavidin-PE reagent (SAPE, 6 µg/ml) and incubated 20 min at room temperature, with shaking. Following SAPE incubation, the beads were washed 3 times in Wash Buffer 2, resuspended in xMAP Sheath Fluid, and analyzed in a Luminex200 plate reader.
Size exclusion chromatography
EVs from cell culture conditioned media were concentrated in a 100kDa MWCO Amicon spin filter (EMD Millipore, Cat. No. UFC910096), and plasma EVs were concentrated by precipitation with an optimized PEG reagent (NeuroDex). The concentrated samples (0.5 and 2 ml, as appropriate) were subjected to size exclusion chromatography (SEC) using pre-calibrated columns (IZON Science, qEV Original, 35 nm and qEV2, 35 nm for conditioned media and plasma, respectively). Void volume and up to 25 fractions (0.5 and 2.0 ml, respectively) were collected, and protein concentration (A280 absorption) was measured using NanoDrop2000 spectrophotometer (Thermo Fisher Scientific).
EV isolation from conditioned media
Media conditioned by induced pluripotent stem cell (iPSC)-derived neurons was purchased from BrainXell. iPSCs had been differentiated into cortical neurons using BrainXell proprietary protocol. Serum-free conditioned media (120 ml total) were collected over 4 weeks, with 50% of the media collected each week. HEK293 cells were grown in a medium supplemented with 10% FBS; at 50–70% confluence, the media was replaced with EV-free, serum-free basal media, and the cells were incubated for additional 48 hours. The media were cleared by two centrifugation rounds (10 min, 3,000xg), and EV were collected by ion exchange chromatography (IEC) on Q Sephadex as described previously (Kosanovic et al. 2017). The unbound material was washed with excess equilibration buffer (0.05 M Tris–HCl, pH 7.6), followed by step elution in 0.05 M Tris–HCl, pH 7.6, supplemented with 0.25M NaCl to remove weakly bound proteins and with 0.5 M NaCl for total EV elution. Finally, the EV were concentrated by dialysis against NeuroDex storage buffer supplemented with protease and phosphatase inhibitors (Thermo Fisher Scientific, Cat. No 78429, 78426) in 100 kDa MWCO spin filters.
Transmission Electron Microscopy
EV suspensions (four ml per sample, fresh or fixed overnight, in 1% paraformaldehyde) were loaded onto glow-discharged copper mesh Formvar-coated carbon grids (Sigma-Aldrich, Cat. No G4776) and allowed to adsorb for ~ 30s. The grids were briefly washed in ddH2O, stained with 2.5% aqueous Uranyl Acetate solution, and allowed to fully dry on air before imaging. Grids were imaged using a FEI Morgagni transmission electron microscope (FEI, Hillsboro, OR) operating at 80 kV and equipped with a Nanosprint5 CMOS camera (AMT, Woburn, MA).
Detergent treatment of crude EV fractions
Crude EV fractions generated by PEG-based precipitation (plasma EVs) or by centrifugation/ultrafiltration through 100 kDa MWCO Amicon spin filter (Millipore UFC901008) were supplemented with Triton X-100 to a final 2% w/v concentration and incubated for 1–2 hours at room temperature or for 30 min at 50oC.
RNA isolation and amplification
For RNA isolation, SEC fractions 2–5 and 9–14 were pooled and concentrated using 100kDa MWCO Amicon filters. RNA was isolated with the miRNeasy serum/plasma kit (Qiagen, Cat. No 217184) following the manufacturer’s instructions with minor modifications (RNase-free DNase treatment). cDNA was generated using SuperScript™ VILO™ cDNA Synthesis Kit (Thermo Fisher Scientific, Cat. No: 11754050) following manufacturer’s instructions. For mRNA TaqMan kit and primers (Thermo Fisher Scientific, Cat. No. 44-445-56 and 4331182) see Table 3.
Lipidomic profiling was performed by Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MSMS). Lipid extracts were prepared from live microsomes by a modified Bligh and Dyer method (Bligh and Dyer 1959), spiked with appropriate internal standards, and analyzed using Agilent 1260 Infinity HPLC in an Agilent 6490A QQQ mass spectrometer controlled by MassHunter V 7.0 (Agilent Technologies, Santa Clara, CA). Glycerophospholipids and sphingolipids were separated by normal-phase HPLC as described previously (Chan et al. 2012), using an Agilent Zorbax Rx-Sil column (2.1 x 100 mm, 1.8 µm) at 25°C. In a combination of mobile phase A (chloroform: methanol: ammonium hydroxide, 89.9:10:0.1, v/v) and mobile phase B (chloroform: methanol: water: ammonium hydroxide, 55:39:5.9:0.1, v/v) phase A was maintained at 95% for 2 min, decreased linearly to 30% over 18 min, further decreased to 25% over 3 min, then restored to 95% over 2 min and maintained for 6 min. Separation of sterols and glycerolipids was carried out on a reverse-phase Agilent Zorbax Eclipse XDB-C18 column (4.6 x 100 mm, 3.5mm) with an isocratic mobile phase, chloroform, methanol, 0.1 M ammonium acetate (25:25:1) at a flow rate of 300 µl/min.
Lipid species were quantified by multiple reaction monitoring (MRM) transitions (Hsu et al. 2004; Guan et al. 2007; Chan et al. 2012) using positive and negative ionization modes together with referencing of appropriate internal standards: PA 14:0/14:0, PC 14:0/14:0, PE 14:0/14:0, PG 15:0/15:0, PI 17:0/20:4, PS 14:0/14:0, BMP 14:0/14:0, APG 14:0/14:0, LPC 17:0, LPE 14:0, LPI 13:0, Cer d18:1/17:0, SM d18:1/12:0, dhSM d18:0/12:0, GalCer d18:1/12:0, GluCer d18:1/12:0, LacCer d18:1/12:0, D7-cholesterol, CE 17:0, MG 17:0, 4ME 16:0 diether DG, D5-TG 16:0/18:0/16:0 (Avanti Polar Lipids, Alabaster, AL). To calculate lipid content for each sample, the sums of the number of moles of all species measured by all three LC-MS methodologies were normalized to mol %. The final data are presented as mean mol % ± S.E.M.