Amicon tube

PPP was centrifuged at 12,000× g for 5 min at 4 °C, and 150 μL of the supernatant were loaded onto qEV single columns (70 nm, Izon, Lyon, France). The columns were used according to the manufacturer’s instructions. Six hundred microliters EV fraction and 4 mL protein fraction were collected and subsequently concentrated using Amicon tubes (10 kDa, Merck, Darmstadt, Germany) according to the recommendations of the manufacturer. As soon as volumes were below 200 μL, the sample was collected and diluted to exactly 200 μL with nuclease-free water (NFW).

Culture supernatants or bioreactor harvests were filtered using 0.22 μm bottle top filters (TPP, Trasadingen, Switzerland). For purification, 1 mL KappaSelect columns (GE Healthcare, Little Chalfont, United Kingdom) were used with an ÄKTAPrime (GE Healthcare) or fast protein liquid chromatography (FPLC) system. Phosphate-buffered saline (PBS) served as running and washing buffer, and the flow rate was set to 1 mL/min. Chromatography was carried out at ambient temperature while supernatants were kept on ice during sample loading. Antibodies were eluted with 10 CV 0.1 M glycine pH 2.5, and 1 mL fractions were collected in 1.5 mL centrifuge tubes containing 200 μL 2.45 M potassium phosphate buffer pH 7.2 for neutralization. Fractions containing protein according to UV absorbance at 280 nm were pooled. Buffer exchange of the solution into PBS was carried out by concentrating and diluting using 50,000 molecular weight cut off Amicon tubes (Merck Millipore, Billerica, MA, USA). For adequate buffer exchange, this step was repeated at least 7 times, followed by 0.22 μm filtration using a syringe filter. Protein concentration was determined using a NanoDrop 2000c (Thermo Fisher Scientific, Waltham, MA, USA) spectrophotometer. Antibodies were stored at 4 °C.

NH₂-SiO₂ NPs (500 µg/mL) were mixed with CXCL5 (4 µM) (Peprotech, Rocky Hill, NJ, USA) in water, then 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) (Fluorochem Ltd., Hadfield, UK) 10 µM was added, and the reaction was stirred at room temperature for 4 h. The solution was then centrifugated twice (1100 rpm for 1.5 min) using 100 kDa Amicon tubes (Merck Millipore, Burlington, MA, USA), and the NPs were dispersed in water at the same initial concentration.

J-Lat cells were exposed to NTP, and cell-free supernatants were collected by centrifugation 24 h post-incubation. Supernatants were concentrated 6-fold in 10 kDA Amicon tubes (MilliporeSigma) and protein concentrations were measured by the Bradford protein assay using the Pierce protein assay kit (ThermoFisher Scientific, 23225) following manufacturer’s protocol. Released HMGB1 protein was detected using a recombinant rabbit monoclonal antibody (ThermoFisher Scientific, MA5-31967) diluted in TBS-Tween-20 with 1% BSA and anti-rabbit HRP secondary antibody diluted in TBS-Tween-20 supplemented with 10 g milk powder. Total protein was quantified using chemiluminescence detection with the SuperSignal West Dura procedure as previously described by the manufacturer (ThermoFisher Scientific), and protein bands were quantified using ImageJ.

The polyclonal IgGs from serum or plasma were purified by incubating with equal volume of Protein A and G Sepharose resin (GE Healthcare) at 4°C overnight, followed by washing with PBS and elution of IgGs with 2 M citric acid into 0.2 M TrisBase. Eluents were buffer-exchanged into PBS using 30-kDa Amicon tube protein concentrators (MilliporeSigma). The purified polyclonal IgGs were then digested into Fabs using the Pierce Fab Preparation Kit (Thermo Fisher Scientific). Briefly, after preparation of the IgG sample using Zeba Spin Desalting Columns, 0.25 ml of equilibrated immobilized papain was incubated with 1 to 2 mg of IgG in digestion buffer rotating for 4 to 5 hours at 37°C. Digest was separated from immobilized papain by centrifugation, followed by mixing incubation with Protein A resin at room temperature for 30 min. The flow-through after centrifugation containing Fabs was then buffer-exchanged into tris-buffered saline using 10-kDa filter Amicon tubes (MilliporeSigma). For EMPEM, 500 μg of serum Fab was added to 15 μg of Env trimer and incubated overnight at 4°C. The EMPEM complex was purified from excess Fab using size exclusion chromatography. The complex peak was concentrated to about 0.03 mg/ml and deposited directly onto an nsEM grid.

The presence of glycans in rpS3 was determined using peptide-N-glycosidase F (PNGase F) and concanavalin A lectin. For treatment with PNGase F (NEB, USA), the supernatant was concentrated 15-fold using Amicon tubes with a molecular weight cutoff of 10 kDa (Millipore, USA). For the immunoprecipitation assay, antibody to rpS3 (2 μg) was added to the supernatant and incubated on a rocking platform for 2 hr at 4°C. Protein-A agarose beads were added and incubated for an additional 16 hr at 4°C. After incubation, the samples were washed four times with DPBS. The purified rpS3 was pre-denatured in 1 × glycoprotein denaturing buffer at 100°C for 10 min, and the denatured proteins were treated with PNGase F in a mixture with 10 × G7 buffer and 10% NP-40 at 37°C for 2 hr according to the manufacturer's recommendations. The digested proteins were analyzed by immunoblot after separation on large (21 cm × 19 cm) 12% SDS-PAGE.
Concanavalin A (Con A) was used to assess the sugar composition. Glycoproteins in the cell lysates and concentrated cell culture media were isolated using a Con A-based glycoprotein isolation kit (Thermo Scientific, USA). Con-A bound proteins were subjected to immunoblotting.