To begin, take EVs isolated from Calu6 cells, transfected with fluorophore-conjugated EV-micro RNA, and cell-micro RNA. Dilute the EVs in an appropriate volume of ultra-filtered PBS to prepare the extracellular vesicle suspension for flow cytometry analysis. Use the nano-flow cytometer to analyze nano-sized particles.
Launch the software and configure the instrument according to the recommended calibration instructions. Using performance nano-beads, conduct the performance test. The instrument's performance is deemed optimal if the nano-beads are detected within the standard size range.
Determine the suitable flow rate for EV particle detection. Now, perform a quality control check using ultrapure filtered water to verify the instruments fluidics and assess the performance of detectors and lasers. Configure the parameters to evaluate the sensitivity and resolution of different populations in the bead mix as per the manufacturer's protocol.
Then, apply appropriate gating to distinguish the bead population from instrument noise. Next, thoroughly vortex the samples to ensure the even distribution of EVs before loading them for analysis. Launch the flow cytometry data analysis software and introduce ultra-filtered PBS.
Using ultra-filtered PBS, set up gating for EV particles ensuring that the gating corresponds to the appropriate size of particles based on the calibration. Take EVs that have been transferred and vortexed in an attuned compatible tube and load the tube into the instrument. Capture EV signals while plotting FSC on the x-axis and SSC on the y-axis.
For cell micro RNA detection on the x-axis, select fluorescence signal 1, and for EV micro RNA detection on the y-axis, choose fluorescence signal 2. Using the EV samples isolated from cells transfected with a single micro RNA. Established compensation parameters for the fluorophores.
Flow cytometry revealed a time dependent accumulation of miR-451a, Alexa fluor-488 in EVs supporting efficient packaging and release. In contrast, the fluorescence of cell miR-67 was not observed in EVs.
