U87EGFRvIII cells were grown in 15-cm plates (~20 million cells/plate) in 20 ml media containing 5% EV-depleted FBS. For EV depletion, FBS was ultracentrifuged (100,000 × g) for 16 h at 4°C and the supernatant filtered under sterile conditions using a 0.22 µm filter (Millex-GV, PVDF; Millipore, Billerica, MA). Conditioned media was collected after 48 h and processed as follows: 300 × g speed spin at 4°C for 10 min, followed by transferring the supernatant to a clean Falcon tube and centrifuging again at 2,000 × g. The supernatant was removed and re-centrifuged at 10,000 × g for 30 min; the remaining pellet was resuspended in 100 µl of PBS and stored on ice for 1 h. This is labelled “large EVs’. The supernatant was transferred to Beckman polyallomer tubes and ultracentrifuged at 100,000 × g for 1 h at 4°C. The pellet containing predominantly small EVs was resuspended in 100 µl of PBS and labelled “small EVs”. A fixed angle rotor 70 Ti was used to isolate EVs from conditioned media. A MLA-55 fixed angle rotor was used to isolate EVs from serum of glioma-bearing mice.
Beckman polyallomer tubes
Manufactured by Beckman Coulter
Beckman polyallomer tubes are laboratory centrifuge tubes made of a durable polymer material. They are designed for use in a variety of centrifugation applications.
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Lab products found in correlation
2 protocols using beckman polyallomer tubes
1
Isolation of Large and Small EVs
2
Chlamydospore Purification from Candida albicans
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Radiating C. albicans colonies on agar plates were removed using a sterile blade and suspended in 3M sodium thiocyanate in TE buffer. The C. albicans laden agar pieces were incubated for 10 min at 50°C with intermittent vortexing to solubilize the agar. After centrifugation at 4,000 rpm for 5 min, the C. albicans cell-hypha pellet was resuspended in PBS buffer, and chlamydospores were separated from suspensor cells by sonication (30 s sonication and 30 s resting cycles for 5 min on ice).
Chlamydospores were purified on linear sucrose density gradients [48 (link)] of 35–66% w/w (1.15–1.32 g/cc), which were prepared by layering successive 2 ml fractions of decreasing density in 15 ml Beckman polyallomer tubes, whereupon 1–2 ml of sample in 35% sucrose was layered on top. Centrifugation was carried out at 39000 rpm in a SW41 swinging bucket rotor for 12 hours at 10°C in a L70 Beckman ultracentrifuge.
Chlamydospores were purified on linear sucrose density gradients [48 (link)] of 35–66% w/w (1.15–1.32 g/cc), which were prepared by layering successive 2 ml fractions of decreasing density in 15 ml Beckman polyallomer tubes, whereupon 1–2 ml of sample in 35% sucrose was layered on top. Centrifugation was carried out at 39000 rpm in a SW41 swinging bucket rotor for 12 hours at 10°C in a L70 Beckman ultracentrifuge.
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