Parental and mutant strains were cultivated in yeast extract–peptone–dextrose (YPD, 5 ml) medium for 24 h (30°C, with shaking). The cell suspensions were adjusted to 3.5 × 107 cells/ml and 300 μl were inoculated onto YPD agar plates (n = 3). The cultures were incubated to confluence for 24 h at 30°C.
The cells were gently recovered from each plate with an inoculation loop and suspended in PBS (30 ml). For removal of the cells and debris, the suspensions were first centrifuged at 5000 × g for 15 min at 4°C, and the resulting supernatants were centrifuged at 15 000 × g for 15 min at 4°C. The supernatants were filtered through 0.45-μm pore syringe filters and centrifuged at 100 000 × g for 1 h at 4°C to recover EVs. EVs were analyzed by nanoparticle tracking analysis (NTA) on an LM10 nanoparticle analysis system, coupled with a 488-nm laser and equipped with an SCMOS camera and a syringe pump (Malvern Panalytical, Malvern, United Kingdom), as previously published (Reis et al. 2021 (link)). Data were acquired and analyzed using the NTA 3.0 software (Malvern Panalytical).
The cells were gently recovered from each plate with an inoculation loop and suspended in PBS (30 ml). For removal of the cells and debris, the suspensions were first centrifuged at 5000 × g for 15 min at 4°C, and the resulting supernatants were centrifuged at 15 000 × g for 15 min at 4°C. The supernatants were filtered through 0.45-μm pore syringe filters and centrifuged at 100 000 × g for 1 h at 4°C to recover EVs. EVs were analyzed by nanoparticle tracking analysis (NTA) on an LM10 nanoparticle analysis system, coupled with a 488-nm laser and equipped with an SCMOS camera and a syringe pump (Malvern Panalytical, Malvern, United Kingdom), as previously published (Reis et al. 2021 (link)). Data were acquired and analyzed using the NTA 3.0 software (Malvern Panalytical).
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