Avanti j 26 xp centrifuge

The pET-21b(+)-NemR^C106 plasmid^{44 (link)}, which contains only one cysteine (Cys106), was transformed in E. coli BL21 (DE3) cells. Cells were grown in LB supplemented with 50 µg/ml of kanamycin at 37 °C until the A₆₀₀ reached 0.8. IPTG (0.5 mM) was used for the expression induction, followed by 3 h of incubation at 37 °C. Harvested cells were then pelleted at 4 °C, 5000 rpm for 15 min using the Avanti^® J-26xp centrifuge (Beckman Coulter^®) with a JLA-8.1000 rotor and resuspended in lysis buffer composed of 50 mM Tris/HCl pH 8, 0.2 M NaCl, 1 mM DTT, 0.1 mg/ml AEBSF, 1 μg/ml Leupeptin, 50 μg/ml Dnase I, and 20 mM MgCl₂. Cells were disrupted and centrifuged as mentioned above. The supernatant was in-batch incubated with Ni²⁺-Sepharose 6 Fast Flow beads (Cytiva) equilibrated with 50 mM Tris/HCl pH 8, 0.2 M NaCl and 1 mM DTT for 1 h at 4 °C. The beads were packed in a column, and the AKTA™ Pure system (GE Healthcare, Life Sciences) controlled by UNICORN 6.3.0.731 software was used for purification. NemR^C106 was eluted using a linear gradient with elution buffer consisting of 50 mM Tris/HCl pH 8, 0.2 M NaCl, 1 mM DTT and 0 to 700 mM (0–100%) imidazole. Following purification, protein purity was assessed on a non-reducing SDS-PAGE gel, and the pure fractions were dialyzed (~20 ml sample/2L dialysis buffer) overnight at 4 °C against the binding buffer and stored at −20 °C.

Expression of TetA(B)-His10 and TetA(B)-Δ394–401-Thrombin-His12 constructs were optimized in E. coli strain C43 [45,46] . Fresh transformations of each construct were made and plated on to 90 mm 2 × TY 1.5% (w/v) agar plates containing 100 μg/ml ampicillin. Colonies were picked into 10 ml of 2 × TY containing 100 μg/ml ampicillin and grown for 18 h in sterile test tubes at 37 °C and 250 rpm. 4 ml of this bacterial culture was used to inoculate 1 l of 2 × TY containing 100 μg/ml ampicillin in a 2 l conical flask, which was incubated at 37 °C, shaking at 220 rpm in an Infors HT Multitron standard shaker until the cell density reached an OD₆₀₀ of 0.3–0.5. IPTG was added to a final concentration of 0.2 mM and the temperature was lowered to 27 °C for 4 h. Cells were harvested in 1 l centrifuge bottles (Beckman Coulter) at 3063 ×g for 15 min at 4 °C in a JLA-8.1000 rotor (Beckman Coulter) in Avanti J-26 XP centrifuge (Beckman Coulter) and resuspended in 20 ml per litre of cells of 20 mM Tris pH 7.4 with protease inhibitor tablets made up to manufacturer's specifications, which were then frozen at − 20 °C in 50 ml Falcon tubes.

Cell cycle separation was conducted using an elutriation system with JE-5.0 elutriator rotor (Beckmann Coulter Inc) equipped with Avanti J-26 XP centrifuge (Beckmann Coulter Inc) and a pump. 2X10^8 cells were harvested and washed twice with Elutriation buffer (3.4mM EDTA and 1% FBS in 1X PBS). In order to obtain a single cell suspension the cell pellet was resuspended in 40ml Elutriation buffer and passed twice through an 18-gauge needle (25G) syringe. Next, the sample was loaded into the pre-assembled elutriation chamber. Throughout the elutriation process the centrifuge was maintained at a constant speed of 1700rpm at 4°C. To obtain elutriation fractions, the flow rate of the elutriation buffer was increased from 8ml/min to 20ml/min by 1ml/min increments. Consecutively 200ml effluent volumes were collected from the centrifuge for each flow rate and cells were pelleted by centrifugation. To assess the quality of synchronization in each elutriated fractions, cells were stained PI followed by FACS analysis. Based on the cell cycle profile similarity, elutriation fractions were further grouped and categorised into 3 different fractions.

hCMEC/D3 were cultured in triple flasks (Thermo Fisher Scientific). Media were collected after exposure of cells to the different experimental conditions and centrifuged at 2,000 g for 15 min at 4 °C, followed by centrifugation at 10,000g for 45 min at 4 °C (5810R centrifuge, Eppendorf, Hamburg, Germany). Supernatants were filtered through a 0.22 µm filter (Sartorius, Göttingen, Germany) and supplemented with NaCl at a final concentration of 75 mM and polyethylene glycol-6000 (PEG; Sigma-Aldrich) at a final concentration of 10%. sEVs were concentrated at 1,500 g for 30 min at 4 °C (Avanti J-26 XP centrifuge, Beckmann Coulter, Brea, CA, U.S.A.). Pellets were then dissolved in 0.9% NaCl (Sigma-Aldrich), transferred to ultra-clear centrifuge bottles (Beckmann Coulter) and precipitated by ultracentrifugation at 110,000g for 130 min at 4 °C (Optima L7-65, k factor: 133, Beckmann Coulter). sEV pellets were resuspended in 0.9% NaCl supplemented with 10 mM HEPES (Life Technologies) and stored in low retention microcentrifuge tubes (Kisker Biotech, Steinfurt, Germany) at -80 °C until further use.

The tag-free P-VP8* proteins were expressed in Escherichia coli (E. coli) (strain BL21, DE3) induced by 0.4 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at room temperature (~25 °C) overnight [17 (link),43 (link)]. For purification, clarified bacterial lyses were treated with ammonium sulfate [(NH₄)₂SO₄] at 1.0 M end-concentrations for 30 min to precipitate the P-VP8* proteins. After centrifugation at 5000 rpm for 20 min with an Avanti J26XP centrifuge (Beckman Coulter, Brea, CA, USA) using a JA-17 rotor, the protein pellets were washed twice using 1 M (NH₄)₂SO₄ solution in PBS. The P-VP8* protein precipitations were then solved in 20 mM Tris buffer (pH 7.5). The proteins were further purified by an anion exchange chromatography and then analyzed by gel-filtration chromatography (see below). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze protein quality using 10% separating gels. It was also used to quantitate proteins using serially diluted bovine serum albumin (BSA, Bio-Rad, Hercules, CA, USA) with known concentrations as standards on the same gels [15 (link)].

Fruit samples from the four parental lines and from the progeny of the three mapping populations were partially thawed and homogenized in a food processor (CombiMax 700, Braun GmbH, Kronberg, Germany). Phenolic compounds were extracted from duplicate aliquots (10 g) with methanol (20 ml) by homogenization in a Polytron, PT3100 homogenizer (Kinematica AG, Littau, Switzerland) at 28,000 rpm for 30 s. The extracts were centrifuged at 39,200 × g for 10 min at 20 °C (Avanti J-26 XP Centrifuge, Beckman Coulter, USA), following which the supernatants were collected and the insoluble plant material was re-extracted as above with 70% methanol (20 ml). The two pooled supernatants of each sample were combined, and the volume was made up to 50 ml with 70% methanol and stored at −80 °C until analyzed.