Medium derived from both HGC27-R and KATOIII-R was processed for exosome isolation, in according to a previously published procedure (27 (link)) and as reported in the MISEV 2018 guidelines (28 (link)). Briefly, the culture medium of cells was centrifuged for 10 minutes at 1500× g (Thermo Scientific, Heraeus Multifuge X3 Centrifuge). The supernatant was transferred to a clean tube and centrifuged again at 1800× g for 10 min. Subsequently, the supernatant was carefully transferred into a sterile tube and further centrifuged first at 3000× g for 15 minutes and then at 3800× g for 15 minutes. The super-natant was ultra‐centrifuged at 75,000× g for 2 hours (BECKMAN, L‐60 Ultracentrifuge) and, the supernatant again ultra‐centrifuged at 100,000× g for 2.5 hrs. All centrifugation steps were carried out at 4 °C. The pellet consisting of purified exosomes was recovered and dispersed in 200 μL of ultrapure water for the chemical-physical characterization and protein extraction or in sterile PBS for the cultured cell. After the exosome extraction, a characterization of freshly isolated exosome samples was performed by Dynamic Light Scattering (DLS) analysis, ς-Potential measurements and Transmission Electron Microscopy (TEM) investigation as previously reported (27 (link)). The remaining sample was stored at -80°C until protein extraction was assessed.
Heraeus multifuge x 3 centrifuge
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
The Heraeus™ Multifuge™ X 3 Centrifuge is a high-performance laboratory centrifuge designed for a variety of applications. It features a microprocessor-controlled system and can achieve a maximum speed of 15,000 rpm with a maximum relative centrifugal force (RCF) of 21,382 x g. The centrifuge is equipped with a large-capacity rotor that can accommodate a range of sample containers, including microtubes, falcon tubes, and blood collection tubes.
Automatically generated - may contain errors
3 protocols using heraeus multifuge x 3 centrifuge
1
Exosome Isolation from Cell Culture
2
Listeria monocytogenes Reference Strains
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Three different commonly used reference strains of L. monocytogenes (LO28, EGD-e and 10403S) were used in this study. LO28 is a serotype 1/2c, while EGD-e and 10403S both belong to serotype 1/2a. All bacterial strains were kept in cryovials supplemented with 7.0 % (vol/vol) dimethyl sulfoxide (DMSO; Sigma-Aldrich, Dorset, UK) at -80 o C. Before experiments, strains were streaked onto Brain Heart Infusion (BHI) agar (LABM, Lancashire UK) and incubated overnight at 37 o C. A single colony from each plate was transferred with a sterile loop in 3 mL of BHI (LAB M, Lancashire UK) and incubated overnight at 37 o C without shaking. To prepare the final working cultures, 20 mL sterile BHI were inoculated with the overnight culture and left for incubation at 37 o C for 24 h without shaking. Cultures from stationary phase were harvested by centrifugation (Heraeus™ Multifuge™ X 3 Centrifuge, Thermo Scientific, UK) at 9000 ×g for 5 min (room temperature), washed twice using sterile Phosphate-buffered saline (PBS pH 7.1; Oxoid, Basingstoke, UK) and re-suspended in the same medium. Further serial decimal dilutions were prepared for direct plating on BHI agar. The plates were incubated for 24 h at 37°C and viable cell number was expressed as log CFU/mL.
3
Listeria monocytogenes Reference Strains
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Three different commonly used reference strains of L. monocytogenes (LO28, EGD-e and 10403S) were used in this study. LO28 is a serotype 1/2c, while EGD-e and 10403S both belong to serotype 1/2a. All bacterial strains were kept in cryovials supplemented with 7.0 % (vol/vol) dimethyl sulfoxide (DMSO; Sigma-Aldrich, Dorset, UK) at -80 o C. Before experiments, strains were streaked onto Brain Heart Infusion (BHI) agar (LABM, Lancashire UK) and incubated overnight at 37 o C. A single colony from each plate was transferred with a sterile loop in 3 mL of BHI (LAB M, Lancashire UK) and incubated overnight at 37 o C without shaking. To prepare the final working cultures, 20 mL sterile BHI were inoculated with the overnight culture and left for incubation at 37 o C for 24 h without shaking. Cultures from stationary phase were harvested by centrifugation (Heraeus™ Multifuge™ X 3 Centrifuge, Thermo Scientific, UK) at 9000 ×g for 5 min (room temperature), washed twice using sterile Phosphate-buffered saline (PBS pH 7.1; Oxoid, Basingstoke, UK) and re-suspended in the same medium. Further serial decimal dilutions were prepared for direct plating on BHI agar. The plates were incubated for 24 h at 37°C and viable cell number was expressed as log CFU/mL.
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