Anti mouse alexa647

10⁷ EVs derived from each cell line were diluted in PBS in a final volume of 50 µL. Anti-human CD9 (1:100; Cymbus Biotechnology, London, UK) and anti-human CD63 (1:100; clone H5C6, BD Pharmingen, California, USA) were added and samples were incubated for 1 h at RT and, after that, anti-mouse IgG FITC or anti-mouse Alexa 647 (1:200; ThermoFisher Scientific, Waltham, MA) were added and incubation proceeded for 1 h at RT. For staining control, EVs were incubated only with secondary antibody. 1 mL of filtered PBS was added to each sample and the fluorescent intensity were determined using CytoFLEX flow cytometry (Beckman Coulter, CA, USA). The equipment was calibrated using a mixture of fluorescent beads with size ranging from 100 nm to 1 µm for vesicles detection. Data analysis was performed with CytExpert 2.0 Software.

Neurons expressing A-Ch or SA-Ch were fixed in 2% formaldehyde 5% sucrose phosphate-buffered saline (PBS) and permeabilized in 0.1% Triton X-100. After PBS washing, samples were blocked in 1% bovine serum albumin (BSA) PBS, and primary antibodies anti-Cherry (GeneTex GTX59788, 1:500) and anti-PSD95 (Abcam ab9909, 1:600) were used in 0.5% BSA PBS. After washing, primary antibodies were detected with anti-rabbit-TRITC (Sigma-Aldrich T6778, 1:200) and anti-mouse-Alexa647 (Thermo Fisher A32728, 1:200) in 0.5% BSA PBS. Coverslips were mounted in Fluoroshield (Sigma-Aldrich) mounting medium. Hippocampal neurons expressing EGFP, ChETA/EGFP, S-Ch/EGFP, or SA-Ch/EGFP for 24 h were processed as above. Primary antibody was 1:2,500 anti-MAP2 (Abcam ab5392) and it was detected with anti-chicken-Alexa647 (Abcam ab150171, 1:250). For surface NMDAR/AMPAR immunostaining, div 9 neurons were transfected with SA-Ch and palmitoyl-Turquoise2, or palmitoyl-Turquoise2 alone; on the third day from transfection, neurons were fixed in 4% formaldehyde, 5% sucrose PBS and washed, blocked in 5% BSA PBS, and stained with 1:500 anti-GluR1-NT (Millipore MAB2263) and 1:500 anti-GluN1 (Alomone AGC-001), and followed by 1:200 anti-mouse-Alexa488 (Thermo Fisher A32723)/1:200 anti-rabbit-Alexa647 (Thermo Fisher A32733) and mounting.

Following PBS washes cells were permeabilised with 0.1% (v/v) Triton X-100 for 5 min and then washed in PBS and blocked for 30 min in block buffer (1% BSA, 2% goat serum in PBS). Tubulin was immunolabelled with 2 µg ml⁻¹ anti-α-tubulin (Clone DM1A, Sigma-Aldrich) diluted in block buffer for 1 h at room temperature. Tubulin was secondary labeled with 4 µg ml⁻¹ anti-mouse-Alexa647 and F-actin was labeled with 13 nM phalloidin-Alexa488 (Thermo-Fisher). Cells were washed in PBS and mounted in Hydromount (National Diagnostics) prior to imaging.