Optiprep density gradient

The seawater samples for the vesicle analysis were collected from Station ALOHA on cruise HOT263 (June of 2014). At each depth, we collected 100 to 175 L by using a Niskin bottle array, and we concentrated the <0.2 μm fraction via tangential flow filtration, following previously described protocols (1 (link)). The vesicles were enriched across an Optiprep density gradient (Iodixanol; Sigma-Aldrich), as previously described (1 (link)). The field particle concentrations were extrapolated from Nanosight measurements of the concentrated, gradient-purified fractions that contained the highest abundance of vesicles (between approximately 1.14 to 1.19 g/mL). We note that particle losses are incurred during the sample processing and analysis pipeline. Thus, these in situ measurements should be considered to be lower-bound estimates. The cellular abundance, PAR, and temperature values were obtained from the Hawaii Ocean Time-series program (https://hahana.soest.hawaii.edu/hot/hot-dogs/interface.html). As vesicle-like particles could be produced by microbes from all domains of life, the cell abundances represent the combined counts for cyanobacteria, heterotrophic bacteria, and eukaryotes from the indicated depths.

Experiments were performed on adult C57 black mice (4-8 weeks of age) according to the guidelines of the animal care and use committee of East Carolina University, an AAALAC-accredited facility. Mixed cortical/hippocampal neurons were isolated and cultured with minor modifications [31 (link)]. In brief, cells were released from dissected hippocampi and cortex by incubation with papain (Worthington Biochemical Corporation, Lakewood, NJ, USA) by gentle trituration. Subsequently, neuronal fractions from the cells were separated utilizing OptiPrep density gradient (Sigma, St. Louis, Mo, USA) and seeded in Neurobasal medium containing B27 supplement, 0.5 mM glutaMax, gentamycin, mouse FGF2, and PDGFbb (10 ng/mL) growth factors (Thermofisher Scientific, Waltham, MA, USA) onto 35 mm poly-D-lysine coated glass bottom dishes (MatTek, Ashland, MA,USA) and incubated at 37°C. Upon neurite and synaptic formation (7-10 DIV), cells were transfected with glycosylated (wt) and unglycosylated (N220/9Q) forms of Kv3.1b using Lipofectamine^® 2000 reagent (Thermofisher Scientific, Waltham, MA, USA) as described [18 (link)]. Cells greater than 48 h post transfections were utilized for live cell total internal reflection fluorescence (TIRF) microscopy studies, and to make whole cell lysates for western blots.

Bacillus subtilis was spread in an agar plate containing 3% Trypticase soy broth (TSB), 0.5% Yeast extract (YE), and 1.5% Bacto Agar (all from BD Biosciences, San Diego, CA, United States) and incubated at 37°C for 9 h. One colony was picked and inoculated in 25 ml of 3% TSB and 0.5% YE liquid media. Then, it was incubated for 5 h in the shaking incubator at 150 rpm at 37°C until the OD value reached between 0.45 and 0.6. For sporulation, culture was transferred to 500 ml of the autoclaved media containing 5 ml of 10% KCl, 5 ml of 1.2% MgSO₄⋅7H₂O (pH 7.6), 0.5 ml of 1 M Ca(NO₂)₃, 0.01 M MnCl₂, and 1 mM FeSO₄. The culture was incubated at 37°C for 48 h with shaking at 150 rpm. The cells were collected by centrifugation at 5516 × g for 10 min, re-suspended in distilled water, and incubated at 4°C for 48 h on the rocker. Then, the cells were sonicated at 35% amplitude (1 watt) for 90 s with 0.5 s pulse. Spore loaded on the layers of 35, 25, and 15% OptiPrep Density gradient (Sigma-Aldrich, Wt. Louis, MO, United States) was centrifuged at 10,000 × g for 40 min at 25°C without break for the purification. The spore was washed 3 times with distilled water and re-suspended in 1 ml of distilled water.

Exosomes were isolated from cell culture supernatants by differential centrifugation. RA synovial fibroblasts were plated at 200,000 cells/well in a 6-well tissue culture plate in serum-free media. Media was exchanged and incubated for 24 h after which the supernatant was collected. An aliquot of the supernatant was taken for later analysis and the remaining amount was subjected to several steps of differential centrifugation. All centrifugation was conducted at 4 °C. First, the supernatant was centrifuged at 300 × g for 10 min to remove free cells. The remaining supernatant was centrifuged at 10,000 × g to remove cellular debris, then at 30,000 × g to remove smaller debris. The supernatant was then transferred to an ultracentrifuge and spun at 110,000 × g overnight to pellet the exosomes. The supernatant was stored and the pellet was separated on an Optiprep density gradient (Sigma-Aldrich, St. Louis, MO, USA). The fractions containing exosomes were then isolated. Notably, exosomes isolated from RA and OA SFs were isolated similarly from rheumatoid factor-depleted SFs. The original whole supernatant, exosome and cellular debris-depleted fractions, exosome fractions, exosomes isolated from SFs as well as exosome fractions lysed with 0.5 % Triton X-100 (Sigma-Aldrich) were individually analyzed using the Id1 ELISA.