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19 protocols using normal human serum

1

Complement-Dependent Virus Neutralization Assay

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Complement-dependent neutralization assays were carried out as described previously, with slight modifications [11 (link)]. In total, 3 × 108 pfu of VSV harvested from HeLa and A549 cells at specific time intervals (0–6, 6–12, 12–18 and 18–24 h) were incubated with PBS or Normal Human Serum (NHS) (Complement Technologies, Tyler, TX, USA) at a dilution of 1:1. The virus incubated with PBS only served as the control. After incubation at 37 °C for 30 min, the viral titers were determined by a plaque assay on a monolayer of Vero cells. The reduction in the number of plaques with respect to the input virus represented the virus neutralization. The individual data points represent an average and standard deviation of three independent experiments. Student’s t-test was used for the statistical analysis, and p ≤ 0.05 was considered as significant.
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2

Quantifying Complement Activation Markers

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The levels of anaphylatoxins C3a and C5a were measured as an indicator of complement activation. Cells were incubated with 1:8 diluted normal human serum (NHS) (Complement Technology) which was used as a source of complement. After 4 h of incubation, the conditioned medium was collected and measured for C3a and C5a levels using Complement C3a Human ELISA Kit (Thermo Fisher) and Human Complement C5a ELISA Kit (Abcam), respectively. All measurements were performed in triplicates.
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3

Quantifying Complement Inhibition by Borrelia Lipoproteins

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Inhibition of CP-mediated erythrocyte hemolysis by recombinant B. burgdorferi lipoproteins was assayed using a modified version of the previously described classical pathway hemolytic assay (28, (link)54) (link). Normal human serum (Complement Technologies) was diluted to 2.3% (v/v) in CP/LP reaction buffer. GST-tagged ErpB, ErpQ, BBK32, or BB0460 proteins were serially diluted two-fold, 125 μl of each dilution was mixed with 125 μl of the diluted serum, and the mixtures were incubated at room temperature for one hour. During incubation, 5 ml of preopsonized sheep erythrocytes (Complement Technologies) were centrifuged (400 × g, 3 minutes, 4°C) and washed twice in CP/LP reaction buffer. After washing, erythrocytes were resuspended in 5 ml of CP/LP buffer and 40 μl of the erythrocyte suspension were added to each of the incubated serum-protein mixtures. These reactions were incubated for one hour at room temperature, gently vortexing every 15 minutes to ensure that erythrocytes remained in suspension. Following incubation, samples were centrifuged (600 × g, 3 minutes, 4°C) and 200 μl of supernatant from each sample was collected and the OD405nm was measured in a BioTek Synergy HT plate reader using Gen5 software.
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4

Bacterial Lysis by Complement System

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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.
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5

Complement Activation Evaluation by ELISA

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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)
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6

Culturing and Serum Opsonization of F. alocis

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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.
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7

B. burgdorferi Serum Resistance Assay

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Assays were performed as outlined previously (20 (link), 31 (link), 32 (link)). Briefly, B. burgdorferi strain B314 producing B. hermsii FbpC (pAP2), as well as the vector-only control B314 pBBE22luc, were grown at 32°C in 1% CO2 in complete BSK-II media with kanamycin at 300 μg/ml to early-to mid-log phase. The cells were then diluted in complete BSK-II media to a final cell density of 1×106 cells/mL, then incubated for 1.5 hours in 15% normal human serum (Complement Technology) or equivalent heat inactivated serum as a positive control for survival. Cell survival was assessed by dark field microscopy based on cell motility and membrane damage or lysis.
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8

Quantifying Complement Activation by Nanoparticles

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The sizes of MOG-PLGA-PEG-NP and MOG-PLGA-NP were determined via DLS and were used to calculate the weight of particles needed to achieve a desired total surface area based on a NP density of 1.2 g/cm3 [43 ]. To test for complement activation, 0.0025 m2 of NPs in 10 μL of water were added to 40 μL of normal human serum (Complement Technology, Inc., Tyler, TX) in a 96-well sterile cell-culture plate and incubated at 37 °C for 1 h. As a positive control, mouse IgG (Sigma-Aldrich, St. Louis, MO) was heat-aggregated at 70 °C for 30 min and added to the serum at a final concentration of 1 mg/mL. Zymosan (Complement Technology, Inc., Tyler, TX) was added to serum at a final concentration of 0.5 mg/mL. Complement activation was quenched by adding 12.5 μL of 0.05 M EDTA solution in PBS and the samples were transferred to clean 1.5 mL tubes. NPs were removed by centrifuging at 18,000 × g for 7 min and the supernatants were collected and tested for complement activation. ELISAs were performed using the MicroVue C4d and C5a EIA kits (Quidel, San Diego, CA) according to manufacturer instructions.
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9

Culturing and Labeling of Oral Anaerobes

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F. alocis ATCC 38596 and low passage clinical isolate D-62D were cultured in brain heart infusion (BHI) broth with yeast extract supplemented with L-cysteine (0.1%) and arginine (0.05%) for 14 days anaerobically at 37°C as previously described (Aruni, Roy, & Fletcher, 2011 (link); Moffatt, Whitmore, Griffen, Leys, & Lamont, 2011 (link)). Heat killed F. alocis was generated by incubation at 90 °C for 60 min. Non-viability was confirmed by incubation in culture media at same conditions used for the live organism. Serum opsonization was performed by incubation of F. alocis at 37°C for 20 min in different percentages, 2-5-10%, of normal human serum (Complement Technology, Inc.) For fluorescence immunostaining assays, F. alocis was labeled with carboxyfluorescein succinimidyl ester (CFSE; 40 ng/μl) for 30 min at room temperature in the dark, and washed 3 times with PBS prior to use. Peptoanaerobacter stomatis strain CM2 was cultured anaerobically at 37°C in Tryptic Soy Broth supplemented with 20 g/L yeast extract, 1% hemin and 1% reducing agent (37.5 g/L NH4Cl, 25 g/L MgCl2 × 6H2O, 5 g/L CaCl2 × 2H2O, 50 g/L L-cysteine HCl, 5 g/L FeCl2 × 4H2O) as previously described (Jimenez Flores, Tian, Sizova, Epstein, Lamont, & Uriarte, 2017 (link)).
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10

SH-SY5Y Neuroblastoma Cytotoxicity Assay

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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.
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