Microsep advance centrifugal device

In order to determine whether nanoparticles or high molecular compounds are responsible for the interference, the chosen extract was subjected to three steps of filtration. The stock solution of 5 was filtrated through syringe PTFE (Whatman, Little Chalfont, UK) 13 mm filters with decreasing pore size (450 nm, 200 nm and 100 nm). After each step filtrate was stored and used for bioassays and chromatographic determination.
Removal of high molecular pyrogens from extracts was performed for all samples using ultrafiltration process^{34 (link)}. For this purpose, were used Microsep™ Advance Centrifugal Devices (Pall Corporation, New York, United States). The extracts were centrifuged twice, first using filters with cut-off value 100 kDa, second 30 kDa.

Leaves of 5‐week‐old N. benthamiana plants were inoculated with agrobacterial suspensions diluted to an OD₆₀₀ of 0.15 with infiltration buffer (10 mm MES, 10 mm MgSO₄, 0.1 mm acetosyringone, pH 5.6). Three days after infiltration, the leaves were submerged in extraction buffer (50 mm sodium phosphate/200 mm KCl, pH 7.0) prior to vacuum exposure in a desiccator. Exogenous buffer was removed prior to centrifugation of the leaves for 15 min at 2000 g and 4 °C. The recovered exudate was then supplemented with 20 mm imidazole and loaded on a 1 mL column of Chelating Sepharose (GE Healthcare) charged with Ni²⁺ ions. After washing with the same buffer, bound proteins were eluted with 250 mm imidazole in extraction buffer. Protein‐containing eluate fractions were pooled, dialysed twice against 2 L of extraction buffer and then concentrated by ultrafiltration using Microsep Advance centrifugal devices (molecular weight cut‐off: 10 kDa; Pall Corporation, New York, NY, USA).

The culture of 5H9 hybridoma cells [49 (link)] and the production of mouse monoclonal CD9 Ab were recently described [50 (link)]. The corresponding CD9 Fab was generated using the Pierce Fab Purification kit (#44985, Thermo Fisher Scientific). Herein, CD9 Ab (500 µg) was incubated with papain immobilized on agarose resin for 3 h at 37 °C. The digested Abs were collected by centrifugation (5000× g, 1 min) using a spin column, and the flow through containing the Abs was placed in a new tube. The fragment crystalline (Fc) fragment was removed from digested Ab samples by centrifugation (1000× g, 10 min) using the NAb Protein A Plus Spin Column, and the flow through containing the purified Fab fraction was collected. The column was then washed twice with PBS and each wash fraction was combined with the Fab fraction. Using the Microsep Advance Centrifugal Device (10K molecular weight cut-off) purchased from Pall Corporation (#MCP010C46, Westborough, MA, USA), the Fab fraction was concentrated by spinning at 3000× g for 25 min at 4 °C. Concentration was then measured by absorbance at 280 nm (final yield of 0.4–0.8 mg/mL). CD9 Fab preparation was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie blue staining as described in [50 (link)].

To evaluate the bioactivity of the bacterial culture filtrate against fungal growth, we had initially cultivated both WR5 and M4 bacterial strains in Jensens medium separately. At an O.D. of 0.4, the culture was diluted to 10⁻³X in the Hill-Kaefer broth and cultured for three days at 28 ± 2 °C. Cells were harvested by centrifugation (13,000 rpm for 20 min at 4 °C), and supernatants were filter sterilized through 0.22 μm filter (Millex, 33 mm, Millipore). After filter sterilization, the culture filtrates were used for mycelium growth assay in three day-old broth culture containing mycelium in the culture flask by adding 1% culture-filtrates per day for eight days. For plate assay, filter sterilized bacterial supernatants were again passed through the 1kDa cut-off Microsep advance centrifugal device (Pall Corporation) for isolation of active bacterial metabolites. The ultra-filtrates were concentrated up to 20-fold by vacuum evaporation at 37 °C. The concentrated metabolites were used for fungal growth assay in petri dishes by adding 20 μl volume into each disk in the three day-old culture on Hill and Kaefer agar plates, which was continued till eight days.