HEK293T cells were washed with PBS and transferred to 1.5 ml tubes. The cells were counted and 106 cells pelleted by centrifugation at 400 x g for 3 min at 4oC. The cell-pellet was resuspended in 200 μl RIPA (Thermo) buffer and incubated for 20 min on ice, followed by sonication using a Biorupter (Diagenode) (7 cycles of 30 s with 30 s pause between cycles) at 4oC. The same number of cells was analyzed as input control. The cell lysate was then transferred to 0.5 ml open-top thickwall polycarbonate tubes (Beckman, #343776) and centrifuged at 100.000 x g for 1h at 4oC in a TLA-120.1 rotor (Beckman, #362224). The supernatant was transferred to a 1.5 ml tube and the pellet was resuspended in 250 μl 1x HU buffer (8 M urea, 5% SDS, 200 mM Tris-HCL pH 6.8, 1 mM EDTA, 0.01% bromphenol blue, 2% β-mercaptoethanol). The supernatant and input were TCA-precipitated and resuspended in 250 μl 1x HU buffer for subsequent immunoblot analysis.
Open top thickwall polycarbonate tubes
Manufactured by Beckman Coulter
Sourced in United States
Open-top thickwall polycarbonate tubes are laboratory equipment designed for general use in various experimental procedures. They feature a simple, open-top design and are constructed from durable polycarbonate material.
Automatically generated - may contain errors
Lab products found in correlation
Tla 120.1 rotor, by Beckman Coulter (1 mentions)
Biorupter, by Diagenode (1 mentions)
Radioimmunoprecipitation assay (ripa), by Thermo Fisher Scientific (1 mentions)
Optima max, by Beckman Coulter (1 mentions)
Ripa lysis buffer, by Cell Signaling Technology (1 mentions)
Optima max ultracentrifuge, by Beckman Coulter (1 mentions)
3 protocols using open top thickwall polycarbonate tubes
1
Fractionation and Analysis of Cellular Lysates
2
Ultracentrifugation of Extracellular Vesicles
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The labelled EV suspension was diluted to a 1 ml final volume with DPBS. Two different centrifugation parameters were used in accordance with the parameters used for the initial EV isolation: the 20k EVs were pelleted with centrifugation at +4 °C with 20 000×g (18 185 rpm, k-factor 289) for 1 h, and the 110k EVs were pelleted with centrifugation at +4 °C with 110 000×g (42 647 rpm, k-factor 53.0) for 2 h. The centrifugations were done by an Optima MAX ultracentrifuge equipped with the MLA-130 fixed angle rotor in 1 ml Open-Top Thickwall polycarbonate tubes (Beckman Coulter, USA). The supernatant was aspirated just above the formed pellet to avoid disturbing the rather loose pellet, and the EVs were allowed to disperse in 70 μl of DPBS overnight at +4 °C.
3
Isolation of Extracellular Vesicles from Plasma
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Extracellular vesicles were isolated according to standard protocols from our group [26 (link), 27 (link)]. Briefly, plasma aliquots were thawed and each 500 µl were diluted into 1 mL of 1X PBS and centrifuged at 3,000 × g for 20 min at 4 °C and later at 10,000 × g for 30 min at 4 °C to remove cell debris. Supernatants were recovered, diluted with 1X PBS, and centrifuged at 100,000 × g for 70 min at 4 °C into 6.5 mL, Open-Top Thickwall Polycarbonate Tubes (Beckman Coulter) in an Optima MAX Ultracentrifuge (Beckman Coulter). Finally, EVs pellets were resuspended in 1X PBS or lysed with 1X RIPA lysis buffer (Cell Signaling) and sonicated for 2 min for further analysis.
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