Dapi fluoromount

Confocal analysis was performed to visualize if EVs were incorporated by MCF-7 and MCF7-Cx46 recipient cells, confocal analysis was performed. Image stacks were obtained from MCF-7 and MCF-7Cx46-GFP cells incubated with EVs derived from high expression MCF-7Cx46-GFP (EVs-Cx46) and EVs derived from low expression MCF-7 cells (EVs-sans-Cx46). MCF-7 cells were incubated for 3 h at 37 °C or 4 °C with EVs-Cx46 and EVs-sans-Cx46. After that, cells were washed with 4 °C PBS to eliminate non-incorporated EVs, fixed, and permeabilized with Triton X-100 (0.2% v/v), incubated with CellMask Deep Red (which strains the entire cell) (Thermofisher, Waltham, MA, USA) at 1× final concentration for 30 min, washed with PBS and mounted using Dapi-Fluoromount (Electron Microscopy Sciences, Hatfield, USA), and visualized in an FV1200 Olympus confocal microscopy. Image were acquired in a 60x AN1.4 oil immersion objective at 2048 × 2048 pixel resolution with a Z step of 10 × 0.5 μm using the corresponding excitation and emission settings for; PKH26 (EVs) (551ex, 567em), Cell Mask Deep Red (stains the entire cell) (Thermofisher, Waltham, MA, USA) (649ex, 666em), and GFP (Cx46-GFP) (450ex, 480em). Z-stack projections were obtained and intensity correlation analysis was carried out using the Fiji software (NHI, Bethesda, MD, USA) [49 (link),50 (link)].

Cells were grown to confluency on either untreated 18 mm glass coverslips (Electron Microscopy Sciences; for HeLa cells) or collagen-coated 18 mm glass coverslips (Neuvitro; for SH-SY5Y cells). After any treatments, cells were washed twice in PBS and then fixed in 4% paraformaldehyde (PFA; Electron Microscopy Sciences). Fixed cells were permeabilized in 0.1% Triton X-100 (Thermo Fisher) for 15 min at RT and then blocked in 5% bovine serum albumin (BSA; Gemini Bio-Products) for 40 min at RT. Coverslips were incubated in primary antibodies (Supplementary Table S3) diluted in 1% BSA for 1 h at RT and then washed twice in PBS + 0.2% tween (PBST) and once in PBS and incubated in secondary antibodies (Supplementary Table S3) diluted in 1% BSA for 1 h at RT. Coverslips were washed twice in PBST and once in PBS before being quickly rinsed in deionized water. Coverslips were mounted to glass slides with DAPI-Fluoromount (Electron Microscopy Sciences) and cured overnight. Images were taken with a Zeiss LSM800 microscope with a Plan-Apochromat 63x/1.4 NA oil lens and processed with ImageJ (NIH).