Differential ultracentrifugation was performed as described previously by Théry et al. [9 (link)] with a few modifications. Briefly, 1 mL of embryo-conditioned medium was diluted to 5 mL in phosphate-buffered saline (PBS) (Invitrogen), transferred to a 5.2-mL open-top polyallomer centrifuge tube (Beckman Coulter, Fullerton, CA, USA) and centrifuged for 7 min at 10,000× g and 4 °C in a swinging bucket centrifuge (Optima XPN-80, SW 55 Ti rotor, Beckman Coulter). The pellet was discarded, and the supernatant was centrifuged for 30 min at 30,000× g and 4 °C, again the pellet was discarded, and the supernatant was centrifuged for 3 h at 100,000× g and 4 °C. The pellet was resuspended in 100 µL of PBS and stored at −80 °C for further EV characterization.
5.2 ml open top polyallomer centrifuge tube
Manufactured by Beckman Coulter
Sourced in United States
The 5.2-mL open-top polyallomer centrifuge tube is a laboratory equipment item used for centrifugation. It is designed to hold a volume of 5.2 milliliters. The tube is made of polyallomer material.
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2 protocols using 5.2 ml open top polyallomer centrifuge tube
1
Isolation of Extracellular Vesicles via Differential Ultracentrifugation
2
Extracellular Vesicle Isolation from Conditioned Medium
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One millilitre of CCM was diluted to 5 mL in phosphate buffered saline (PBS) (Invitrogen), transferred to a 5.2 mL open top polyallomer centrifuge tube (Beckman Coulter, Fullerton, CA) and centrifuged for 3 hours at 100,000 g and 4°C in a swinging bucket centrifuge (Optima XPN-80, SW 55 Ti rotor, Beckman Coulter). The pellet was resuspended in 50 µL of PBS and stored at −80°C. Note: (a) in this protocol the 10,000 g. Hence it has no meaning anymore in this section. Centrifugation step was omitted due to the prior use of 0.45 and 0.2 µm filters to obtain the CCM, and (b) no extra washing step was carried out in regard to recently published observations (11 ).
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