Express plus

Dulbecco's modified Eagle's medium with EV‐depleted FBS was prepared by ultracentrifugation of culture medium for 16 hours at 110 000 g overnight at 4°C using a SW28 rotor followed by filtration through a 0.22 µm filter (Millipore Express PLUS (PES) membrane). EVs were isolated from conditioned media (CM) of cultured NPCE cell line or from CM of Primary NPCE cells using a polyethylene glycol (PEG) based approach as previously described.30, 31 Supernatant was collected from cells that were grown in medium containing EV‐depleted FBS for 48 hours and was subjected to centrifugation steps at 300 g for 10 minutes and 2000 g for 10 minutes to remove cell debris and larger vesicles. The resulting supernatant was filtered using 0.2 µm filter and added to the filtered precipitation solution (50% PEG8000 (Sigma), 0.5 mol/L NaCl in PBS), to achieve final PEG concentration (8.3% w/v), and samples were then mixed by repeated inversion and incubated overnight at 4°C. EVs were precipitated by centrifugation at 1500 g for 30 minutes, and the supernatant was removed. Residual fluid centrifugation was eliminated by centrifugation at 1500 g for 5 minutes and pelleted EVs were resuspended in PBS for further analysis.

HS-(CH₂)₁₁-EG₃-OH (hPEG) and HS-(CH₂)₁₁-EG₃-OCH₃ (mPEG) were purchased from Prochimia (Sopot, Poland). (3-aminopropyl)-trymethoxysilane was purchased from Gelest, Inc. (Morrisville, PA). Solution P: 18 mg/ml phenylmethylsulfonyl fluoride (PMSF), 0.3 mg/ml pepstatin A in ethanol, cOmplete EDTA-free protease inhibitor cocktail (PIC) (Roche Applied Science). All other chemicals were purchased from Sigma-Aldrich Co. (Saint Louis, MO), as ethylenediaminetetraacetic acid (EDTA), sodium dodecyl sulfate (SDS), 2-mercaptoethanol (βME), tris-(2-carboxyethyl)phosphine (TCEP). The buffer solutions prepared for the experimental work were: (i) PB: 0.01 M phosphate buffer, pH 7.4; (ii) PB-E: PB and 0.01 M EDTA; (iii) PBS: PB and 2.7 mM KCl and 137 mM NaCl₂, pH 7.4; (iv) PBS-ET: PBS, 10 mM EDTA and 5 mM TCEP; (v) TB: 20 mM HEPES, 110 mM KOAc, 2 mM MgCl₂, 10 μM CaCl₂ and 10 μM ZnCl₂, pH 7.4; (vi) TBT: TB and 0.1% v/v Tween 20; (vii) TBT-D: TBT and 5 mM DTT; (viii) TBT-PVP: TBT and 0.3% (w/v) polyvinylpyrrolidone; (ix) HUT: 50 mM HEPES, 8 M urea and 0.5% Tween 20. All buffers were prepared fresh and filtered through a 0.22 μm polyethersulfone membrane Express Plus from Millipore (Billerica, MA). Compressed N₂, He and O₂ were supplied by Airgas (Albany, NY).

Various transfected IgG1 antibodies, FuG1 antibodies, and recombinant IgG4-Fc antigens (as indicated in text and figure legends) were affinity purified using HiTrap MabSelect SuRe (GE, 11003493) protein-A columns. Transfected cultures were harvested after 10 days and filtered through 0.2-micron PES membrane filters (Milipore Express Plus). Cleaning-in-place (CIP) was performed for each column using 0.2M NaOH wash (20 min). Following cleaning, columns were washed three times with Binding buffer (20 mM sodium phosphate, 0.15 M NaCl, pH 7.2). Filtered supernatant containing recombinant antibodies or antigens were passed through the columns at 4°C. Prior to elution in 0.1M sodium citrate, pH 3.0 to 3.6, the columns were washed three times with binding buffer (pH 7.0). The pH of eluted antibodies was immediately neutralized using sodium acetate (3M, pH 9.0). After protein measurements at 280 nm, antibodies were dialyzed in phosphate-buffered saline (PBS) using Slide-A-Lyzer 3.5K (Thermo Scientific, 66330). Antibodies were run on gel filtration columns (see next section) to analyze the percent monomers. Whenever necessary a second step size exclusion chromatography (SEC) was performed. Recombinants IgG4-Fc tagged extracellular domain antigens such as rFOLR1, rDR5, and RBD, etc. were also similarly harvested and purified using protein-A columns.

Various transfected IgG1 antibodies, FuG1 antibodies, and recombinant IgG4-Fc antigens (as indicated in text and figure legends) were affinity purified using HiTrap MabSelect SuRe (GE, 11003493) protein-A columns. Transfected cultures were harvested after 10 days and filtered through 0.2-micron PES membrane filters (Milipore Express Plus). Cleaning-in-place (CIP) was performed for each column using 0.2M NaOH wash (20 min). Following cleaning, columns were washed three times with Binding buffer (20 mM sodium phosphate, 0.15 M NaCl, pH 7.2). Filtered supernatant containing recombinant antibodies or antigens were passed through the columns at 4°C. Prior to elution in 0.1M sodium citrate, pH 3.0 to 3.6, the columns were washed three times with binding buffer (pH 7.0). The pH of eluted antibodies was immediately neutralized using sodium acetate (3M, pH 9.0). After protein measurements at 280 nm, antibodies were dialyzed in phosphate-buffered saline (PBS) using Slide-A-Lyzer 3.5K (Thermo Scientific, 66330). Antibodies were run on gel filtration columns (see next section) to analyze the percent monomers. Whenever necessary a second step size exclusion chromatography (SEC) was performed. Recombinants IgG4-Fc tagged extracellular domain antigens such as rFOLR1, rDR5, and RBD, etc. were also similarly harvested and purified using protein-A columns.

Water samples were collected from 10 m depth at stations H3 (36°44.34N, 122°01.20W; coastal) and 67-70 (36°07.56N, 123°29.46W; offshore) in Monterey Bay, CA, USA on the 1^st and 9^th of October 2009, respectively. Samples were immediately 0.22 μm filtered (Millipore Express Plus, Millipore, MA, US) and stored at 4°C in the dark in acid-washed polycarbonate bottles until further analysis. Nutrient measurements were conducted as previously described [43 (link)].