Millipore express plus

Suspension cultured media were filtered using a 0.22 µm Millipore Express^® PLUS membrane filter (Millipore, Volketswil, Switzerland). His-tagged rrhGAAs in the filtrate were purified using Ni-NTA Superflow Cartridges (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocols. Confirmation of purified rrhGAA was performed by SDS-PAGE analysis (Fig. S2).

The bacterial EVs were isolated by a procedure described earlier (Askarian et al., 2018 (link); Wagner et al., 2018 (link)), with slight modifications. A fresh overnight culture of the methicillin-susceptible S. aureus MSSA476 (1:100 dilution) was inoculated into 500 mL BHI (normal conditions) or Chelex-treated BHI broth containing 0.5 μg/mL of vancomycin (stressed conditions) at least two to five different days. The cultures were grown with shaking at 37°C for 16 h. The cultures were then centrifuged at 6,000 × g for 30 min. Bacterial pellets were discarded, and the supernatants was filtered through 0.2 μm filters (Millipore Express™ Plus, USA) and ultra-centrifuged at 100,000 × g for 3 h at 4°C in a 45 Ti rotor (Beckman, USA). EV pellets from each isolation were re-suspended in 500 μL RNAlater (Thermo Fisher Scientific, Massachusetts, USA) or in Phosphate-buffered saline (PBS) if EVs were to be used for microscopy or Nanoparticle Tracking Analysis, and kept at −80°C until further use. Prior to RNA isolation for RNA-seq, EVs from several isolations were thawed, pooled and concentrated using ultrafiltration (10 kDa Vivaspin 20, Sartorius, Germany). An overview of EV isolations from bacteria grown under stressed conditions and its downstream applications is provided in Supplementary Table 1.

Filtration and autoclaving methods were used for the sterilization of lead NE and NEM formulations. Sterilization of the MOX-NE and MOX-NEM formulations by filtration technique was investigated through 0.22 µm filter pore size. Samples (1.0 mL) were passed through various filter membranes using a 13 mm stainless-steel Swinny filter holder (MilliporeSigma, St. Louis, MO, USA). Different filter membranes were tested, such as Millex^® syringe filters with nylon membranes (0.20 µm), Durapore™ (PVDF; polyvinylidene difluoride, 0.20 µm), Fluoropore™ (PTFE; polytetrafluoroethylene, 0.22 µm), and Millipore Express^® PLUS (PES; polyethersulfone, 0.22 µm). The filtrate was collected in a glass vial, and the effect of the filtration technique on the physicochemical characteristics of the MOX-NE and MOX-NEM formulations was evaluated.
MOX-NE and MOX-NEM formulations were also subjected to autoclaving (121 °C under 15 psi for 15 min, 3850ELP-B/L-D Tuttnauer autoclave, Heidolph, Germany) in glass vials affixed with indicator tapes for the sterilization process. The sterilization cycle was confirmed by the color change of the indicator tapes attached to the glass vials. After the moist heat sterilization process, formulations were stored at RF and RT for one month (last time point tested) in the same containers and analyzed in the same way as the pre-autoclaved samples.

Production and purification of violacein was performed as described previously [18 (link),19 (link)]. Briefly Chromobacterium violaceum ATCC 12742 was cultivated overnight (24 h) in nutrient broth (NB) (Difco BD, Detroit, MI, USA) at 30 °C and 250 rpm in a shaking incubator. To extract the pigment, the bacterial cells were harvested via centrifugation (3600× g, 20 min, 4 °C) and violacein was extracted using 95% ethanol (culture volume) by mixing (250 rpm, 6 h, 30 °C). The bacterial cells were then removed by pelleting them (3600× g, 10 min, 4 °C). This procedure was repeated until violacein was completely extracted from the cells.
The extract was then filtered (Steritop 45 mm, 0.22 µm PES membrane, Millipore Express PLUS, Millipore, Burlington, MA, USA) to remove any remaining bacterial cells before being dried in a rotary evaporator (N-1110, EYELA, Bohemia, NY, USA) at 50 °C. The dried violacein was the solubilized in 50% acetone and boiled 40 °C within the rotary evaporator until crystallization of the violacein was visually evident. Crystallization was allowed to continue for 24 h at 20 °C. The violacein crystals were collected by centrifugation (7200× g, 20 min, 20 °C) and washed with purified water before being dried at 60 °C for 48 h. The purified violacein was then solubilized in DMSO (Sigma-Aldrich) and quantified by HPLC method as described previously [20 (link)].

In order to obtain conditioned media (CM), MSC and GIST cells were co-cultured in either the contact co-culture or non-contact co-culture system, as described previously. The contact co-culture method yielded contact conditioned medium (contact CM), while transwell conditioned medium (transwell CM) was obtained using the non-contact co-culture system. Additionally, GIST-CM and MSC-CM were obtained by separately culturing GIST and MSC cells. All CMs were collected every 6 h, centrifuged, and filtered using a vacuum-driven cup apparatus (Millipore Express PLUS, Merck Millipore, Germany), and then stored in a frozen state. Prior to use, the CMs were thawed and mixed with complete culture media at a 10–20% ratio.

The average hydrodynamic diameter (D_h) and polydispersity index (PDI) of all ASMP-Nano formulations were determined by measuring the rate of fluctuations in laser light intensity scattered by the nanomicelles immediately after their preparations using the Dynamic Light Scattering (DLS) analysis (Beckman Coulter® N4 Plus, Beckman Coulter s.r.l., Milan, Italy). Five minutes prior to light-scattering measurements each sample was diluted with ultrapure water previously filtered through 0.45 μm pore size filter (polyethersulfone membrane, Millipore Express PLUS, Merck, Italy). The final concentration was chosen on the basis of the measurement intensity ranging from 5 × 10⁴ to 1 × 10⁶ counts per second (cps). The average size for each ASMP-Nano formulation was obtained on three different samples for which six runs were carried out using an angle of 90° [30 (link)] and run time of 200 s at 20 °C.
The same protocol analysis was performed to characterise the Nano1HA_BFITC-CyA formulation.