Syto rnaselect green

After the final wash (see Duolink^® PLA Fluorescence Protocol), 100 µL Ca²⁺/Mg²⁺ PBS was added to the cells. To label the cell bodies, washed cells were incubated with 500 nM SYTO RNASelect Green (Invitrogen, ThermoFisher Scientific, MA, USA, S32703) in Ca²⁺/Mg²⁺ PBS for 20 min at room temperature, and then washed twice with Ca²⁺/Mg²⁺ PBS for 5 min each. To stain the nuclei, one drop of Hoechst 33342 Ready Flow Reagent (Invitrogen, ThermoFisher Scientific, MA, USA, R37165) was added to each dish and incubated for 5 min without washing. After the staining, the cells were imaged in Ca²⁺/Mg²⁺ PBS using a confocal Leica TCS SP5 laser-scanning microscope (Leica, Wetzlar, Germany) equipped with an HCX PLAPO CS 63Ч1.4 oil immersion lens. The image acquisition parameters were as follows: -

Hoechst fluorescence (DNA staining) with excitation at 405 nm and emission at 414–487 nm;

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RNASelect Green fluorescence, excitation at 488 nm, emission at 495–590 nm;

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Duolink Detection Reagent (Red) fluorescence, excitation at 594 nm, emission at 600–652 nm.

Images were processed using LAS X software (Leica, Wetzlar, Germany).

The freshly isolated astrocyte-derived EVs were diluted in the 0.22 μm-filtered PBS and were then stained under sterile dark conditions with green-RNA-binding, a liposoluble fluorophore SYTO (Syto RNA Select Green, Invitrogen, USA) that is able to cross the EV membrane. Samples were vortexed and bathed at 37 °C in the dark for 30 min before being loaded into the flow cytometer CytoFlex S (Beckman and Coulter, USA) and visualized by the CytExpert software. The cytometer was washed with detergent and water between the EVs samples to eliminate any remaining residue between samples. CytoFLEX was set up to detect microvesicles using Megamix-Plus FSC beads (BioCytex, ref. 7820), this being a mixture of 100 nm, 300 nm, 500 nm, and 900 nm fluorescent beads. The FITC fluorescence of these beads was analyzed using the 488 nm laser and 525 nm fluorescence emission filter. Moreover, the forward scatter (FSC) and side scatter (SSC) signals from 488 nm laser, the 405 nm side scatter (violet side scatter, VSSC), were used to improve the detection of small particles. In the dot plot FITC/VSSC, fluorescent beads were visualized by adjusting the gains of the fluorescence and scatter detectors. These gains were applied to acquire the samples stained with BODIPY (see Additional file 2: Figure S2).