G24 environmental incubator shaker

SSG was maintained on PDA plates. For liquid culture, a 4 mL NB was inoculated with a single colony and incubated on a G24 Environmental Incubator Shaker (New Brunswick Scientific Inc., Edison, NJ, USA) at 180 rpm and 28 °C overnight, then used as a culture stock. For experiments, 150 mL NB or PDB was inoculated with 1 mL of the SSG stock then incubated for 40 h under the same conditions. The culture was centrifuged at 14,210× g for 15 min. Bacterial cells in pellets were resuspended in 200 mL PDB for in vitro assays or 0.01% Tween 20 for plant inoculation studies. Bacterial cell concentration of resultant resuspensions was determined by spreading 100 µL of its dilutions on PDA then counting the emerging colonies after a 2 day incubation at 25 °C. The resultant bacterial cell concentrations ranged from 10⁸ to 10⁹ colony-forming units (cfu) per milliliter. In the meanwhile, supernatant was further passed through a 0.22 μm filter to produce cell-free supernatant (CFS). Resuspended bacterial cells and CFS were evaluated separately for their potential against Cps unless stated otherwise. The resuspended bacterial cells and CFS treatments hereafter were referred to as Cell and CFS, respectively.

For the investigation of IFN-β decomposition kinetics 52.3 μg ml⁻¹ IFN-β solution was prepared in PBS, and the protein concentration was measured for 4 days using the mBCA assay. The absorbance due to the colour reaction was spectrophotometrically analysed at 562 nm in predetermined times.
The prepared BSA-loaded nanoparticles were washed and redispersed in PBS containing 0.03% w/v sodium azide to get a nanoparticle concentration 5–6 mg ml⁻¹. The washed IFN-β-loaded nanoparticles were suspended in human blood plasma (1 mg ml⁻¹). The release medium was replaced after sampling. The samples were mixed by a rotating mixer (Bio RS-24 mini-Rotator, Biosan, Latvia) with vertical rotation at 30 rpm, and incubated at 37 °C in a G24 Environmental Incubator Shaker (New Brunswick Scientific, USA). At predetermined intervals, 0.5 ml of each sample was ultracentrifuged (Beckman Optima Max-E) for 10 min at 40 000×g. The concentration of released IFN-β was determined using ELISA. The BSA concentration was followed by mBCA assay in the supernatant after removal of the nanoparticles by centrifugation, and the IFN-β was analysed in the removed supernatants by ELISA using a multiplate reader.

All of the E. coli isolates were streaked on lysogeny broth [29 (link)] agar plates and the plates were incubated at 37 °C overnight. A single colony was picked using a sterile pipet tip and inoculated in 5 ml LB broth in a 15 ml culture tube. The culture tubes were incubated at 37 °C with shaking (150 rpm) overnight using New Brunswick G24 Environmental Incubator shaker. Genomic DNA was extracted using the Qiagen DNeasy Blood and Tissue kit (Qiagen, Germantown, MD), and plasmid by GenJet mini prep kit (Thermo Fisher Scientific, Waltham, MA) as recommended by the manufacturer.