Optima l 80 xp

A further purification of the isolated phage particles was conducted as preparation for the proteomics measurements. For this purpose, cesium chloride (CsCl) was dissolved in SM buffer creating solutions of different densities (1.3, 1.4, 1.5, 1.6 g/mL). These solutions were subsequently layered into a centrifuge tube with 2 mL per density (with lowest density on top). 2 mL of pre-purified phage solution (in SM buffer) were added on top of this gradient and centrifuged using open Ultra-Clear Tubes (Beckman Instruments Inc., Palo Alto, CA, USA) in a SW 28.1 swinging rotor (Beckman) with a Beckman Coulter Optima L-80XP ultracentrifuge (Beckman Coulter, Brea, CA, USA) at 150,000× g and 4 °C for 2 h.
During this centrifugation, a milky layer of phage particles appeared between the 1.3 g/mL and 1.4 g/mL layer. This milky layer was extracted, washed three times with SM buffer using Amicon Ultra centrifugal filters with 30 kDa cutoff (MerckMillipore, Burlington, MA, USA) and subsequently tested for their phage titer.

We harvested the cell
culture media from the primary cell lines and A549 cells and centrifuged
it at 750×g at 4 °C for 15 min to remove
the detached cells. Then, we centrifuged the collected media at 2000×g at 4 °C for 20 min to remove the microvesicles. We
collected the supernatant and filtered it through 0.45 μm filters.
We subsequently spun it in a Beckman Coulter Optima L-80XP ultracentrifuge
at 10,000×g at 4 °C for 45 min with a Type
SW 32 Ti rotor to remove the apoptotic bodies and cell debris. We
recovered the supernatant and filtered it through 0.22 μm filters.
We then ultracentrifuged it at 100,000×g at
4 °C for 90 min to pellet the EVs. We carefully removed the supernatant
and resuspended the crude EV-containing pellets in an aliquot of PBS,
which we pooled.

A culture of A. baumannii strain 6077/12 was grown to mid-exponential phase in a volume of 500 mL and was infected with 1 mL of previously obtained phage suspension [titer 10⁹ plaque forming units (PFUs)/mL]. After overnight incubation, the lysate was centrifuged in Sorvall RC3B centrifuge at 13,689.2 × g for 30 min in rotor GS3 to pellet bacterial debris. The supernatant was collected and treated with DNaseI and RNase A (Thermo Fisher) for 2 h at 37°C. Afterward, NaCl and polyethylene glycol (PEG 8000; Sigma) were added (to obtain 1 M and 10% final concentrations, respectively), stirred, and incubated overnight at +4°C to precipitate the phages. The precipitate was centrifuged at 13,689.2 × g for 30 min at +4°C (Sorvall RC3B centrifuge, rotor GS3), and the pellet was resuspended in 4 mL of SM buffer. CsCl was added to reach 0.75 g/mL density. The samples were ultracentrifuged at 131,980.8 × g for 24 h in Beckman Coulter Optima L-80 XP ultracentrifuge in rotor SW55 Ti, after which the distinct phage-containing bluish band was visible at the midpoint of the tube. The phages were extracted from tube by using a 22-gauge needle (PrecisionGlide™; Becton Dickinson, Dubline, Ireland) and dialyzed overnight against 2 L of SM buffer at +4°C. Purified phages were filtered through 0.22-μm filters (Filtropur S 0.45; Sarstedt) and used in subsequent experiments.

The retinas from 4–6-week-old wild-type C57BL/6J mice were extracted and digested using the Papain Dissociation System (Worthington Biochemical Corporation, Lakewood, NJ, USA) according to the manufacturer’s protocol. Exosomes were recovered using differential ultracentrifugation as previously described [26 (link)]. In brief, the supernatant received from the digestion process was mixed with cold HBSS and centrifuged at 25,000× RPM for 60 min at 4 °C in a Beckman Coulter Optima L80-XP ultracentrifuge with rotor SW60 Ti. The supernatant was again subjected to sequential ultra-centrifugation at 41,000× RPM for 60 min at 4 °C for 120 min. The final pellet (exosomes) was resuspended in 100 μL PBS and stored at −80 °C until further use (Figure 1A). The size, number, and morphology of exosomes were characterized by nanoparticle tracking analysis (NTA) (NanoSight, Malvern Instruments Ltd., Malvern, UK) and transmission electron microscopy (TEM), respectively (Figure 1B,D). Exosome markers were analyzed by an exosome detection antibody array (Exo-Check, System Biosciences, Palo Alto, CA, USA) according to the manufacturer’s instructions (Figure 1C). Zeta potential of exosomes was measured using ZetaPALS (Brookhaven Instruments, Holtsville, NY, USA) (Figure 1E).