Exocet exosome quantitation kit

5-dodecanoylamino fluorescein (C12-FAM) and fluorescein were purchased from Thermo Fisher Scientific (USA) and Sigma–Aldrich (USA), respectively. Exosome-depleted fetal bovine serum (FBS), ExoQuick-TC exosome precipitation solution and EXOCET exosome Quantitation kit were purchased from System Biosciences (USA). Dulbecco’s modified Eagle’s medium (DMEM), and penicillin/streptomycin were purchased from Gibco BRL (USA). FBS and Macrosep Advance Centrifugal Devices (30 kDa) were purchased from Youngin frontier (Korea) and Pall Corporation (USA), respectively. Aqueous solutions were prepared using ultrapure DNase/RNase-free distilled water (D.W.) purchased from Bioneer. All other chemicals were of analytical grade and used without further purification.

EVs isolated from yCM and aCM were quantified using the ExoCET^® Exosome Quantitation Kit (System Biosciences, Milan, Italy), following the manufacturer’s instructions. Briefly, EVs were lysed, centrifuged at 1500× g for 5 min, and the supernatants were transferred in the microtiter plate for quantification at 405 nm using a Multiskan FC. The standard curve was obtained through plotting the target concentrations versus absorbances. Linear regression analysis was computed, and EV quantifications were calculated and reported with the highest expression set to 1, while the other expression was relative to this value.

An EXOCET exosome quantitation kit (System Biosciences, USA) was used for exosome quantification. This step was performed according to the manufacturer's instructions. The isolated exosomes were mixed with lysis buffer and incubated at 37 °C for 5 min. After 5 min, the samples were vortexed for 15 s and then centrifuged at 1500×g for 5 min. The supernatant was transferred to a new test tube on ice. Next, 50 μL of lysed exosome protein sample was mixed with 50 μL of premix solution (prepared by mixing buffer A and B from the kit), per well. Then, the mixture-containing 96-well plate was incubated at room temperature for 10 min, and the absorbance was measured at 405 nm. The amount of exosome particles was calculated by comparing absorbance results to a standard curve. Bradford and BCA protein assays were performed to quantify the protein concentration of the exosomes. Briefly, we prepared a standard curve using dilutions of bovine serum albumin (BSA). The Bradford reagent was reacted with exosome protein samples for 5 min in darkness. Then, the absorbance was measured at 595 nm and calculated using the standard curve.

MCPs-EVs were isolated from DMEM-cultured MCPs (Gibco; Carlsbad, CA, USA) in medium supplemented with 20% exosome-depleted FBS (Gibco), with 1% penicillin/streptomycin (Gibco), using a commercial EV isolation kit (ExoQuick-TC, System Biosciences, LLC., Palo Alto, CA, USA) according to the manufacturers’ instructions. The EXOCET exosome quantitation kit (System Biosciences, LLC.) was used to quantify MCPs-EVs, and their concentration was adjusted to 1 μg/μL before treatments.
The MCPs-EV morphology was ascertained by transmission electron microscopy (TEM; Electron Microscopy Sciences, Fort Washington, PA, USA) and their characterization was validated based on the expression of one negative and three positive EV markers in Western blotting [11 (link)] (negative marker: GM130 [1:1000; BD Biosciences, San Jose, CA, USA]; Three positive EV markers [1:1000]: CD9 [Abcam, Cambridge, MA, USA), CD63 [NOVUS Biologicals, Littleton, CO, USA], and CD81 [NOVUS Biologicals]).

Relative quantification of exosomes was performed using the EXOCET Exosome Quantitation Kit (System Biosciences). Basic procedure: A standard curve was prepared using exosome standards provided in the kit. Add 20 μL of exosomes suspension to 80 μL lysis Buffer, incubate at 37 °C for 5 min, centrifuged at 1500×g for 5 min, and incubate the supernatant on ice. 50 μL of the reaction solution was added to 50 μL of the supernatant, and the absorbance was measured at 405 nm after 20 min at room temperature. The number of exosomes was calculated from the standard curve.

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.

EVs quantitation was performed using the EXOCET Exosome Quantitation Kit (System Biosciences, SBI) which is an enzymatic, colorimetric assay designed as a direct measurement of esterase activity known to be within exosomes.
Fresh EVs pooled pellets in a concentrated solution (corresponding to a protein concentration of 1–2 μg/μL) were resuspended with kit lysis Buffer at (1:4, v:v) to a total volume of 100 μL per reaction. EVs lysates were incubated at 37°C for 5 min and centrifuged at 1,500 g for 5 min to remove cell debris. 50 μL of transferred supernatants and standards were added to clear microtiter wells and mixed with 50 μL of reaction buffer (buffer A + buffer B) up to a total 100 µL volume in a 96 well plate (Nunclon Delta, Thermo Scientific). Replicates of each sample were made fourfold, and the microtiter plate was incubated for 20 min at room temperature. Optical density was read using a spectrophotometric plate reader (Apollo 11 LB913, Berthold Technologies GmbH & Co., TN, United States) at 405 nm. Finally, quantitate results were obtained by calculating the standard curve, previously calibrated by Nano Sight analysis, and plotting the sample readings on the standard curve. Quantitative results were represented in number of particles per mL.