Avanti j 20 xp centrifuge

Extracellular vesicles were isolated from transgenic plants and examined for miRNA content. In brief, vesicles were isolated from the apoplastic wash of the vector control and transgenic miR-146a Arabidopsis lines as previously described by Dr. Roger W. Innes [17 (link)]. Whole rosettes were harvested at the root and vacuum infiltrated with vesicle isolation buffer (20 mm MES, 2 mmCaCl2, and 0.1 m NaCl, pH 6). Infiltrated plants were blotted to remove excess fluid, placed inside 30-mL syringes, and centrifuged in 50-mL conical tubes at 700 g for 20 min at 4 °C (JA-14 rotor, Avanti J-20 XP centrifuge; Beckman Coulter). The apoplastic wash was filtered through a 0.22-μm membrane and centrifuged successively at 10,000 g for 30 min, and 40,000gfor 60 min at 4 °C. The vesicle pellet was resuspended in Tris-HCl buffer (10 mM, pH 7.5).

For high-level expression of genes of interest from the trc promoter in E. coli, 500 μl of an overnight culture was inoculated into 500-ml baffled shake flasks containing 100 ml LB medium supplemented with 100 μg ml⁻¹ ampicillin. Cultures were grown aerobically at 37°C in an orbital shaker (Infors, Basel, Switzerland) set at 180 rpm. At an OD₆₀₀ of 0.6, protein expression was induced by addition of isopropyl-β-d-1-thiogalactopyranoside at a final concentration of 200 μM. After 4 h of incubation, cells were harvested, centrifuged (6,500 × g, 15 min, 4°C) in an Avanti J-20 XP centrifuge (Beckman Coulter), washed twice with 100 mM cold Tris-HCl buffer (pH 7.5 at 25°C), and stored at −20°C until further use. BL21 pTrc99a was used as an empty vector control strain.

A. baumannii AB41 and Pseudomonas PAO1 naturally secrete MVs into media. MVs from both strains were collected from broth cultures (500 ml) in the late log phase using an adaptation of the method described by McBroom and coworkers [23 (link)]. The cells were pelleted by centrifugation at 10,000 × g for 10 min at 4°C, and the supernatant was filtered through 0.45-μm-pore-size filters to remove remaining bacterial cells. MVs were obtained by centrifugation at 40,000 × g for 1 h at 4°C in an Avanti J-20 XP centrifuge (Beckman Coulter, Inc.). Pelleted vesicles were resuspended in 50 ml of 50 mM HEPES pH 6.8 (Sigma) and filtered through 0.22-μm-pore-size Ultrafree spin filters (Millipore). Vesicles were again pelleted and finally resuspended in an adequate volume of 50 mM HEPES, pH 6.8 (Sigma). MVs from N. gonorrhoeae were collected from confluent solid cultures grown on CHOC plates. Cells and MVs from 20 agar plates were resuspended in 15 ml of Ringer ¼ (Sigma) per plate and from this moment the MVs were obtained as described for liquid media cultures.
For proteomic studies, MVs from N. gonorrhoeae were further purified by ultracentrifugation in OptiPrep gradients as described by Pérez-Cruz et al. [22 (link)].