Extracellular Vesicle Isolation from Candida Biofilms

Medium portions collected after every 24 h of biofilm culture were pooled and EVs were then isolated using a previously described protocol (Karkowska-Kuleta et al., 2020 (link)) with minor modifications. Briefly, supernatants from cultures of C. glabrata, C. tropicalis, and C. parapsilosis were centrifuged twice at 4,000g for 15 min at 4°C, each time discarding the cell pellet, and then concentrated using an Amicon Ultra-15 Centrifugal Filter Unit with a 100-kDa cutoff (Merck, Darmstadt, Germany) with the addition of cOmplete Protease Inhibitor Cocktail (Roche, Basel, Switzerland). After centrifugation for 5 min at 5,000g and discarding the pellet, the concentrated medium was filtered using an Ultrafree-CL Centrifugal Filter with a pore size of 0.65 µm (Merck) and ultracentrifuged in an Optima™ L-90K Ultracentrifuge (Beckman Coulter, Brea, CA, USA) at 4°C for 1 h at a rotor speed of 45,000 rpm, which corresponds to a relative centrifugal field of 184,000g (k factor 85), using a fixed-angle type 50.2 Ti Rotor and polycarbonate thick wall centrifuge tubes (13 × 64 mm) with 13-mm-diameter Delrin tube adapters. After washing with PBS buffer once, the ultracentrifugation step was repeated and the obtained EVs were transferred in 400 µl of PBS to Eppendorf tubes and stored at −80°C.