To characterize EVs in detail, they purified collected EVs using ultrafiltration combined with size-exclusion chromatography to separate EVs from extrinsic proteins, and then used a nano-flow cytometer (N30 Nanoflow Analyzer, NanoFCM Inc., Xiamen, China) to quantify EVs by their size and surface proteins. In this case, we monitored the astrocyte-specific water channel, aquaporin-4 (AQP4), by using an Alexa Flour 488 conjugated monoclonal AQP4 extracellular domain-specific IgG (Figure 1D). We used AQP4 as an indicator for particle numbers and sizes corresponding to astrocytic EVs collected at different stimulation frequencies. In control condition without applied electrical stimulation, the nano-flow cytometry plot shows populations of AQP4-positive EVs of two distinct sizes (Figure 1E).
In this experiment, they found that the size distributions of AQP4-positive EVs are differentially affected by the frequency of electrical stimulation (Figure 2). Stimulation at 2 Hz produces a near uniform distribution of EVs, with the peak of size distribution at ~70 nm, slightly shifted to the right (larger) by comparison with the smaller sized presumptive exosome population collected without electrical stimulation. The major population of AQP4-positive exosomes arising from 2 Hz stimulation was more AQP4-bright than at baseline, supporting that 2 Hz stimulation generated a population of exosomes with increased membrane AQP4. Stimulation at 20 Hz produced fewer EVs of both large and small size than seen in basal conditions, particularly fewer presumptive exosomes. Electrical stimulation at 200 Hz had the opposite effect of 2Hz stimulation. Instead of yielding exosomes, microvesicles-sized EVs dominated (peak size distribution at ~ 170nm) and this population was also enriched in membrane AQP4.