96 well glass bottom microplates

To demonstrate that pEVs purified by LEAP technology were intact and biologically active, their uptake by recipient cells was monitored by fluorescence microscopy using the lipophilic dye, Exoria (Tertel et al., 2022 (link)). LEAP‐isolated pEVs (approximately 10¹¹ particles/mL) were labelled with a final concentration of 2 μM Exoria and incubated at 37°C for 1 h (Law et al., 2021 ). Free dye was removed with Zeba spin columns, 40K MWCO, as per the manufacturer's instructions (Thermo Fisher, San Jose, CA, USA). Normal human dermal fibroblasts (NHDFs; ATCC) were grown on 96 well glass bottom microplates (Greiner Bio‐One, Kremsmünster, Austria). 20 μL of Exoria‐labelled pEVs or Exoria in PBS (control) were added onto cells and incubated at 37°C for 2 h. Supernatants were removed and the cells were washed three times with PBS. Cells were counter‐stained with 10 μM Hoechst 33342 (Thermo Fisher, San Jose, CA, USA) at room temperature in the dark for 10 min. Cells were washed three times with PBS, then 50 μL of Fixation buffer (Biolegend, San Diego, CA, USA) was added, and cells were incubated at room temperature in the dark for 15 min. Cells were washed three times with PBS, then left in PBS for imaging with a confocal microscope (Nikon A1R, inverted Eclipse Ti‐E model. Images analysed using Image J) (Tertel et al., 2022 (link)).

S. cerevisiae cells were imaged in indicated media at room temperature in 96-well glass-bottom microplates (Greiner Bio-One). Images and semi-three-dimensional time-lapse images were acquired using a Dragonfly 500 spinning disk microscope (Andor) attached to an inverted Ti2 microscope stand (Nikon) with a CFI Plan Apo Lambda 60×/1.4 oil immersion objective (Nikon) and a Zyla 4.2 sCMOS camera (Andor). Fluorophores were excited with excitation lasers 405, 488, and 561 nm; emission was collected using 450/50, 525/50, and 600/50 nm bandpass filters. Image processing was performed with Fiji version 2.1.0 (Schindelin et al., 2012 (link)).