Max xp

Anucleate cells were generated by centrifugation with an Optima MAX-XP tabletop ultracentrifuge equipped with a TLA-100 rotor (www.beckmancoulter.com). Briefly, cells in the exponential phase were centrifuged at 25,000 rpm for 10 min at 4°C, and the cells were then suspended in fresh YE medium and recultured at 30°C for 10–20 min before microscopic observation.

EVs pellets were isolated using a series of differential ultracentrifugation (dUC) steps as previously described (20 (link)). Briefly, 1 mL serum samples from each individual were pooled into three pools for HCs and nine pools for patients with CC (10 mL per pool). Pooled samples were subjected to dUC at 800, 2,000, and 12,000 ×g and filtered through 0.22 µm Minisart pore filters (Cat No. 2SRF2-16534K, Millipore, Burlington, MA, USA). The filtered supernatant was then centrifuged at 110,000 ×g using an optimal MAX-XP ultracentrifuge (Beckman Coulter, Brea, CA, USA) with an MLA-55 fixed-angle rotor. All centrifugation steps were performed at 4 °C. The resulting pellets were resuspended in a qEV size exclusion chromatography (SEC) column (Izon Science, Medford, MA, USA). Fractions were collected according to the manufacturer’s protocol. Briefly, the void volume (volume of the mobile phase required to elute a molecule having zero retention in the stationary phase in SEC) was 5 mL. Using 1× phosphate-buffered saline (PBS) as the elution buffer, 550 µL of the pellet solution was loaded on the column. Directly after this, 500 µL per fraction was collected for a total of 20 fractions. Each collected fraction was then centrifuged at 110,000 ×g for 1.5 h to concentrate the small vesicles. The pooled fractions were collected for further analyses.

Vero cells were used to produce and titrate MYXV. The Vero cells were grown in DMEM with 10% FBS and 1X penicillin/streptomycin at 37 °C with 5% CO₂. Subculturing was carried out using trypsin-EDTA and PBS solutions. MYXV was purified and titrated as previously described [53 ]. Vero cells prepared at 1-1.5x10⁵/mL were infected with green fluorescent protein-labeled MYXV and evaluated under a fluorescence microscope (see Figure 1). To determine the titer of MYXV, a microtitration test using a focal assay was conducted as previously reported [53 ]. The process involved preparing logarithmic 10-fold dilutions of MYXV and applying quadruple repetitions for each dilution. After a 48-h incubation period, the tissue culture infective dose ratio was calculated by counting the growth foci of MYXV. Virus purification was performed in an ultracentrifuge (Optima XPN-100 and Max-XP, Beckman Coulter, USA) with 24%-40% sucrose solutions with double and triple gradients created. The virus layer and two consecutive centrifugation protocols were collected at 50,000 x g for 40 min and at 33,000 x g for 40 min. Finally, the pellet obtained by centrifugation at 18,000 rpm was resuspended in PBS.

The CM was collected and centrifuged at 1000g for 10 min to remove cell debris, followed by centrifugation at 2000g for 10 min and 10,000g for 30 min. The supernatant was then ultracentrifuged at 100,000g for 70 min (L-80XP; Beckman Coulter). The exosome-enriched fraction was resuspended with PBS, ultracentrifuged at 100,000g for 70 min (Max-XP; Beckman Coulter), and diluted with 100 μl of PBS. The morphology and size distribution of exosomes were identified by using transmission electron microscopy (H-7650B, Hitachi) and nanosight tracking analysis (NanoSight LM14, Malvern).