Small cellular particles were obtained by isolation using differential centrifugation. An adapted protocol for isolation of extracellular vesicles (Figure 1 ) 49 (link) was followed. The homogenate was centrifuged at 300 g and 4°C for 10 minutes in the centrifuge Centric 260R with rotor RA 6/50 (Domel, Slovenia). Fifty-milliliter conical centrifuge tubes (ref. S.078.02.008.051, Isolab Labor-geräte GmbH, Germany) were used. The procedure was repeated twice. The supernatant of the second centrifugation at 300 g was centrifuged at 4°C and 2000 g for 10 minutes in the centrifuge Centric 400R with rotor RS4/100 (Domel, Slovenia). Fifteen-milliliter conical centrifuge tubes (ref. S.078.02.001.050, Isolab Laborgeräte GmbH, Germany) were used. The procedure was repeated twice. The supernatant was centrifuged at 4°C and 50,000 g for 60 min in Beckman L8-70M ultracentrifuge, rotor SW55Ti (Beckman Coulter, USA). Thin-wall polypropylene centrifuge tubes (ref. 326819, Beckman Coulter, USA) were used. The supernatant (or 300g I pellet in the case of dilution experiment for flow cytometry) was used in the formation of NSHs (Figure 1 ).
Thin wall polypropylene centrifuge tubes
Manufactured by Beckman Coulter
Sourced in United States
Thin-wall polypropylene centrifuge tubes are designed for use in high-speed centrifugation applications. They are made from polypropylene, a durable and chemically resistant material. These tubes provide a precise and consistent volume for accurate sample handling and processing.
Automatically generated - may contain errors
4 protocols using thin wall polypropylene centrifuge tubes
1
Isolation of Extracellular Vesicles via Differential Centrifugation
2
Isolation of Small Extracellular Vesicles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SCPs were isolated by differential centrifugation, using a protocol widely used for the isolation of small extracellular vesicles [63 (link)]. Briefly, the cells were removed by low-speed centrifugation (300× g, 10 min, 4 °C, centrifuge Centric 260R with rotor RA 6/50 (Domel, Železniki, Slovenia)), using 50 mL conical centrifuge tubes (ref. S.078.02.008.050, Isolab Laborgeräte GmbH, Eschau, Germany); and 2000× g, 10 min, 4 °C (Centric 400R centrifuge with rotor RS4/100 (Domel, Železniki, Slovenia)), using 15 mL conical centrifuge tubes (ref. S.078.02.001.050, Isolab Laborgeräte GmbH, Eschau, Germany). Each step was repeated twice. Then, the cell-depleted medium was centrifuged twice at 10,000× g and 4 °C for 30 min (Beckman L8-70M ultracentrifuge, rotor SW55Ti (Beckman Coulter, Brea, CA, USA)), using thin-wall polypropylene centrifuge tubes (ref. 326819, Beckman Coulter, Brea, CA, USA) to remove larger cell debris. Finally, NPs were pelleted by ultracentrifugation at 118,000× g and 4 °C for 70 min in the same type of ultracentrifuge and ultracentrifuge tubes. The pellet was resuspended in 50 µL of the initial medium (PBS/ultraclean waste/marine water).
3
Extracellular Vesicle Isolation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SCPs were isolated by differential centrifugation as adapted from the protocol for the isolation of extracellular vesicles (EVs) from [44 (link)]. The filtered homogenate was centrifuged twice at 300× g and 4 °C for 10 min in the centrifuge Centric 260R with rotor RA 6/50 (Domel, Železniki, Slovenia) by using 50 mL conical centrifuge tubes (ref. S.078.02.008.050, Isolab Laborgeräte GmbH, Germany). The supernatant of the second centrifugation was centrifuged twice at 2000× g and 4 °C for 10 min in the centrifuge Centric 400R with rotor RS4/100 (Domel, Železniki, Slovenia), using 15 mL conical centrifuge tubes (ref. S.078.02.001.050, Isolab Laborgeräte GmbH, Eschau, Germany). Then, the supernatant was centrifuged at 10,000× g or 50,000× g and 4 °C for 60 min in Beckman L8-70M ultracentrifuge, rotor SW55Ti (Beckman Coulter, Brea, CA, USA) using thin-wall polypropylene centrifuge tubes (ref. 326819, Beckman Coulter, Brea, CA, USA). The respective isolates were obtained by dissolving pellets in a small amount of solvent (e.g., 80 µL).
4
Protein Precipitation from Conditioned Media
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Conditioned media were collected, and dead cells and debris were removed by centrifugation at 1500× g for 5 min. Supernatants were mixed with 99.9% (v/v) ice-cold molecular grade acetone at a ratio of 1:5 in a 50 mL centrifuge tubes and incubated overnight at −20 °C for protein precipitation. Samples were then subjected to initial centrifugation at 1700× g for 5 min (Haraeus Megafuge 1.0) and the supernatants were collected and further centrifuged at 12,000× g for 10 min at 4 °C to precipitate out the proteins (SW28 rotor in Beckman Coulter OptimaTM L-100XP Ultra centrifuge). For this purpose, thin-wall polypropylene centrifuge tubes (Beckman Coulter) were used. Supernatants were concentrated using a Speed vac concentrator (SAVANT SPD 131 DDA, Thermo Scientific, Waltham, MA, USA) to first remove the more volatile acetone. The protein depleted samples were then freeze dried (Martin Christ Beta 2–8 LD Plus) and stored at −80 °C until further processing and analysis.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!