Bacterial cultures were centrifuged (6000 x g, 15 min) and supernatants were filtered (0.22 mm, Nalgene top filters, Thermo Scientific) at room temperature. Cell-free supernatants were then concentrated in successive centrifugations using Amicon ultrafiltration units (100-fold, cutoff 100 kDa, 2500 x g). The concentrated supernatants were then submitted to a series of 3 ultracentrifugation rounds: (1) the first one to pellet EVs and discard the supernatant containing contaminant proteins (150 000 x g, 120 min, 4°C), (2) the second one for a higher-quality density-based separation, with the application of the resuspended pellets onto the top of a discontinuous sucrose gradient (8%–68%) (100 000 x g, 150 min, 4°C), and (3) a third washing step to eliminate the excess of sucrose from pooled EV-containing fractions (150 000 x g, 120 min, 4°C) (Tartaglia et al. 2018 (link), 2020 (link)). The final samples were then resuspended in TBS buffer (150 mM NaCl; 50 mM Tris-Cl, pH 7.5) and used immediately or stored at 20°C.
Nalgene top filters
Manufactured by Thermo Fisher Scientific
Sourced in United States
Nalgene top filters are laboratory filtration devices designed to facilitate efficient separation of liquids and particles. They are compatible with a variety of Nalgene containers and can be used for various filtration applications in research and industrial settings.
Automatically generated - may contain errors
Lab products found in correlation
5 protocols using nalgene top filters
1
Extracellular Vesicle Isolation Procedure
2
Isolation and Purification of Extracellular Vesicles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were pelleted by the centrifugation (6000 g, 15 min, room temperature) of cultures in UF (500 mL) and the supernatant fraction was filtered using 0.22 μm Nalgene top filters (Thermo Scientific) to remove any remaining bacterial cells. The supernatant was then concentrated 1000 times using Amicon ultrafiltration units with a 100 kDa cut-off point in successive centrifugations at 2500 g. The concentrated suspension of EVs was recovered in TBS buffer (Tris-Buffered Saline, 150 mM NaCl; 50 mM Tris–HCl, pH 7.5) and further purified by size exclusion chromatography (qEV original 70 nm; iZON), as recommended by the manufacturer (Böing et al., 2014 (link)). Briefly, 0.5 mL of EV samples was applied to the top of the chromatographic column, followed by TBS buffer for elution. Then, fractions of 0.5 mL were recovered in separate tubes. Fractions 1–6 were discarded as void, EVs-containing fractions (fractions 7–9) were pooled together and the remaining fractions were discarded due to protein contamination or low EV content.
3
Extracellular Vesicles Isolation and Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cultures were submitted to EVs isolation and purification, as previously described (Tartaglia et al., 2018 (link), 2020 (link)). In brief, for each condition 1 L of bacterial cell culture was centrifuged at 6,000 × g for 15 min and filtered through 0.22 μm Nalgene top filters (Thermo Scientific). Then, the culture supernatant fraction was concentrated around 100-fold using the Amicon ultrafiltration systems (Millipore) with a 100kDa filter, and ultra-centrifuged for 120 min at 150,000 × g to eliminate the soluble proteins. Next, the suspended pellet was applied to a discontinuous sucrose gradient (8–68%) and ultra-centrifuged at 100,000 × g for 150 min. Fractions containing EVs were recovered and washed in TBS (150 mM NaCl; 50 mM Tris-Cl, pH 7.5) for final ultra-centrifugation at 150,000 × g (120 min). At last, EVs were suspended in cold TBS and kept at −80°C until use.
4
Isolation and Purification of Extracellular Vesicles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
EVs were isolated and purified as previously described [44 (link)]. Briefly, 1 L of bacterial cell culture was centrifuged at 6000× g for 15 min and filtered through 0.22 μm Nalgene top filters (Thermo Scientific, Waltham, MA, United States). Then, the culture supernatant fraction was concentrated around 100-fold using the Amicon ultrafiltration systems (Millipore, Burlington, MA, United States) with a 100 kDa filter and ultra-centrifuged for 120 min at 150,000× g to eliminate the soluble proteins. Next, the suspended pellet was applied to a discontinuous sucrose gradient (8–68%) and ultra-centrifuged at 100,000× g for 150 min. The fractions containing EVs were recovered and washed in TBS (150 mM NaCl; 50 mM Tris-Cl, pH 7.5) for final ultra-centrifugation at 150,000× g (120 min). At last, the EVs were suspended in cold TBS and kept at −80 °C until use. The quality of the EV samples (i.e., homogeneity, integrity, and reproducibility) was assessed by transmission electron microscopy (TEM), Nano Tracking Analysis (NTA) and SDS-PAGE.
5
Extracellular Vesicle Purification Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were pelleted by the centrifugation (6000 g, 15 min, room temperature) of cultures in UF (500 mL) and the supernatant fraction was filtered using 0.22 µm Nalgene top filters (Thermo Scientific) to remove any remaining bacterial cells. The supernatant was then concentrated 1000 times using Amicon ultrafiltration units with a 100 kDa cut-off point in successive centrifugations at 2500 g. The concentrated suspension of EVs was recovered in TBS buffer (Tris-Buffered Saline, 150 mM NaCl; 50 mM Tris-HCl, pH 7.5) and further purified by size exclusion chromatography (qEV original 70 nm; iZON), as recommended by the manufacturer (Böing et al., 2014) (link). Briefly, 0.5 mL of EV samples was applied to the top of the chromatographic column, followed by TBS buffer for elution. Then, fractions of 0.5 mL were recovered in separate tubes. Fractions 1-6 were discarded as void, EVscontaining fractions (fractions 7-9) were pooled together and the remaining fractions were discarded due to protein contamination or low EV content.
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!