Mircury exosome serum plasma kit

Exosome isolation was performed using 300 μL of plasma fractions with the miRCURY^® Exosome Serum/Plasma kit (QIAGEN, Valencia, CA, USA). Thawed plasma samples were initially centrifuged at 3000× g for 10 min to remove cells and debris before proceeding with the recommended protocol. This protocol consisted of the addition of a precipitation buffer, which diminishes the hydration of the subcellular particles and allows precipitation of small particles with a low-speed centrifugation step, followed by the incubation of the mixture for 1 h at 4 °C. Subsequently, the sample was centrifuged at 500× g for 5 min to obtain the extracellular vesicle pellet.
For exosome characterization, we utilized plasma samples from 3 controls and 5 epileptic dogs, of which 3 were drug-sensitive (DSE) dogs and 2 were drug-refractory (DRE) dogs. (Supplementary Table S1). The protocol was applied twice to each sample. One of the exosome pellets was resuspended in 100 μL of filtered PBS, from which a 50 μL aliquot was set aside for nano tracking analysis (NTA), and various dilutions were prepared with the remaining volume to optimize transmission electron microscopy (TEM) observation. The other pellet was resuspended in 100 μL of RIPA buffer for further protein marker analysis. All aliquots were stored at −20 °C until needed.

After a 5-h fast, ∼1 mL of blood was obtained from the jugular vein, allowed to clot for 30 min at room temperature (RT), centrifuged and the supernatant was collected as serum. Total exosomes were extracted from 500 µL of serum using miRCURY Exosome Serum/Plasma kit (QIAGEN) according to the manufacturer’s instructions. In brief, an initial spin was performed at 10,000 g (RT) for 10 min for each sample to remove cells and debris. The precipitation reagent was added to the sample volume, according to the manufacturer’s instructions. Mixtures were vortexed and incubated at 4°C for 1 h and then centrifuged at room temperature for 30 min at 1,500 g followed by pellet resuspension in resuspension buffer. All exosomes were stored at −80°C immediately after isolation until further analysis.

Whole blood samples (10 mL) were collected in sterile EDTA Vacutainer tubes (BD, Plymouth, UK) and centrifugated twice at 1000 × g for 10 min at room temperature (RT). Plasma samples were stored at − 80 °C until further processing. The miRCURY Exosome Serum/Plasma Kit (Qiagen, Hilden, Germany) was used to enrich for EVs from 500 μL plasma, according to the manufacturer’s instructions, unless otherwise specified. In short, dead cells and debris (including platelets and fibrin) were cleared with thrombin and centrifugation. Precipitation Buffer was added, samples were incubated overnight at 4 °C and the EV fraction was pelleted by centrifugation. Supernatants were collected and stored for separate analysis. EV enriched pellets were resuspended for further processing.

Plasma samples were thawed and centrifuged at 16,000 x g for 15mins at 4°C to pellet cells, debris and platelets. Extracellular vesicles (EVs) were isolated using the miRCURY^® Exosome Serum/Plasma Kit (Qiagen) or the exoEasy Maxi Kit (Qiagen) according to manufacturer’s instructions. Protein concentrations of isolated EV samples (in µg/ml) were determined using the Pierce BCA Protein Assay Kit (Thermo Fisher) according to manufacturer’s instructions. The number of EVs per 100 µg of protein was quantified using the EXOCET Exosome Quantitation Kit (System Biosciences).
pEV RNA was isolated using the exoRNeasy Serum Plasma Kit (Qiagen), according to manufacturer’s instructions. The miRNeasy Serum/Plasma Spike-In Control (1.6 x 10⁸ copies/µl) was added to samples during RNA isolation. RNA was isolated from equal volumes of plasma samples from healthy controls and HIV patients.

Frozen plasma samples were thawed and EVs were isolated using the commercially available miRCURY Exosome Serum/Plasma Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. Briefly, 600 µL of thawed plasma was incubated with thrombin for defibrination, followed by EV precipitation with 500 µL of the thrombin treated plasma and 0.4 volumes of Precipitation Buffer A. Samples were then incubated at 4 °C for 1 hour and then centrifuged at 500× g for 5 min at 20 °C. The supernatant was then separated and the remaining EV pellet was either re-suspended in 200 µL 1X PBS, and stored at −80 °C for downstream characterization, or re-suspended in 700 µL QIAZOL as per the manufacturer’s protocol for RNA purification using the miRNeasy Mini Kit (QIAGEN, Hilden, Germany).