Normal human serum

In order to determine the ability of the complement system to lyse the bacterial samples, 5 µL of EPEC O26:H11 and EHEC O26:H11 (1 × 10¹³ CFU/mL) were added to the wells of a 96-well culture plate. The plate was then incubated for 16 h at 37 °C with 15 µL of heat-inactivated serum or 15 µL of normal human serum (Complement Technology, Tyler, TX, USA) in the presence or absence of 50 µL of anti-O26 polysaccharide antibodies. Subsequently, the bacterial viability was determined by counting the number of colony-forming units (CFU) according to the methodology described by Baron and coworkers and the methodology outlined by Beck et al. [28 (link),29 (link)]. normal human serum was used as a source of complement.

ELISA-based method to evaluate the activation of the classical complement pathway was adapted from (33, 50). Endotoxin-free ELISA plates were coated with wither RBD or Trimer soluble proteins diluted in endotoxin-free PBS (HyClone) overnight. Plates were blocked with endotoxin-free 2% gelatin solution (Sigma) and incubated with anti-Spike antibodies of interest for 1 hour at +4°C. Plates were washed 3 times with endotoxin-free GVB buffer with Ca²⁺ and Mg²⁺ (GVB++, Complement Technology) and incubated with normal human serum (Complement Technology) diluted to 1.25% in GVB++ buffer for 1.5 hours at +37°C on an orbital shaker. Reaction was stopped by a wash with ice-cold PBS. Cells with deposited complement components were stained with anti-C4 antisera (Complement Technology) and a secondary anti-goat-HRP antibody (SouthernBiotech). Plates were incubated with HRP substrate and a stop solution according to manufacturer’s instructions (ThermoFisher). Optical density was measured on EnSpire Plate Reader (PerkinElmer)

F. alocis ATCC 38596 was cultured in brain heart infusion (BHI) broth supplemented 5 mg/mL yeast extract, L-cysteine (0.05%), and arginine (0.05%) for 7 days anaerobically at 37°C as previously described (20 (link), 37 (link)). Serum opsonization was performed by incubating F. alocis at 37°C for 20 min in 10% normal human serum (Complement Technology, Inc., Tyler, TX, USA). Heat-killed F. alocis was generated by incubation at 90°C for 60 min. Non-viability was confirmed by incubation in culture media at the same conditions used for the live organism.

SH-SY5Y neuroblastoma cells (ATCC, #CRL-2266, Manassas, VA, USA) cells were maintained in DMEM with 10% FBS and 1× Penicillin-Streptomycin and plated on a 0.1% gelatin-coated 10 cm tissue culture plate. Cells at passage numbers 5–10 were used for cytotoxicity assays. For live-cell imaging, the cells were seeded in 24-well plates and maintained up to 7 days before treatment when the cell density was about 70% confluency. For EVs cytotoxicity assays, cells were plated at 20,000/cm² in a 96-well tissue culture plate (BD Falcon #353219, San Jose, CA, USA). EV treatment mixtures included various amounts of EVs, synthetic peptides, 5% Normal Human Serum as a source of complement (Complement Technology Inc, Tyler, TX, USA), 1× RealTime-Glo Annexin V Apoptosis reagents (Promega JA1011, Madison, WI, USA), or 1× cell viability assay reagents (2 mM ethidium homodimer-1 or 5 mg/mL of propidium iodide, Waltham, MA, USA), and culture medium to a total of 100 µL per well. Cells were washed once with a fresh warm culture medium before treatment. The treatment mixture was added to SH-SY5Y cells, and the cytotoxicity was measured by relative luminescence (for apoptosis) or green/red fluorescent signals (for live cells and necrosis) every hour with a plate reader (535/717 nm, Biotek Synergy H4 hybrid reader, software version Gen5 3.09, Santa Clara, CA, USA) for 4 h.