As published previously,62 (link) for sEV isolation, PrCa cells (PC3 and DU145) were plated in 150 mm cell culture dishes (ThermoScientific, 130183) in their respective complete medium, as described above. After 48 hours of incubation at 37°C, cells were washed with PBS and incubated with serum-free medium (complete media devoid of FBS) for the next 48 hours. The sEVs were isolated by high-speed differential ultracentrifugation of the supernatant (SN) collected after 48 hours of serum-starvation. The dead cells and cell debris were spun down from SN at 2000 xg, 4°C for 20 minutes. The SN collected was spun at 10,000 xg, 4°C for 35 minutes. Next, the SN collected without disturbing the 10,000 xg pellet was spun at 100,000 xg, 4°C for 70 minutes; the pellet was washed in 40 mL PBS followed by a second spin at 100,000 xg, 4°C for 70 minutes. The 10,000 xg and 100,000 xg centrifugation were done in a Beckman Type 45Ti rotor using a Beckman L8-70M Ultracentrifuge. The final sEV pellets were resuspended in 100 μL PBS.
150 mm cell culture dishes
Manufactured by Thermo Fisher Scientific
The 150 mm cell culture dishes are circular vessels used for cultivating cells in a laboratory setting. These dishes provide a flat surface area for cell attachment, growth, and observation. The dishes are made of polystyrene material and are sterile, single-use products.
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2 protocols using 150 mm cell culture dishes
1
Isolation of Small Extracellular Vesicles from Prostate Cancer Cells
2
Bacterial Filtration Evaluation Protocol
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Bacteria cells (E. coli) were cultured overnight in LB broth at 37 °C with shaking (180 rpm) until the desired optical density (OD) was reached. For treatment of filter materials with E. coli, first, filter paper was cut into small squares to fit into 150 mm cell culture dishes (ThermoFisher, Waltham, MA). One filter paper was added to each culture dish, and one square of control or treated filter material was placed on top of the filter paper. Each sample material was treated with a 50 μL drop of E. coli (1 × 107 CFU/mL). After 1.5 hours of treatment, the tested filter material was removed and the remaining filter papers were placed in 50 mL sample tubes containing 10 mL of LB broth to incubate for 8 hours at 37 °C with shaking (180 rpm). Following incubation, growth of E. coli was measured using an Implen OD600 DiluPhotometer.
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