Ab207002

BEV and parent cells were prepared as described above except that the fractionation of the BEV samples was performed using a qEV/35 nm series size exclusion chromatography (SEC) column and the fractions were obtained according to manufacturer’s instructions (IZON Science, Lyon, France). BEVs typically elute in 6 fractions of 0.5 mL. DPP-4 assays were performed as described by Beauvais et al. (32 (link)). Briefly, 910 μL of 50 mM Tris HCl buffer (pH 7.5) and 50 μL Ala-Pro-pNA (5 mg/mL in methanol) were added to 40 μL of BEV suspension. To assess enzyme binding specificity, 0.33 μM the DPP-4 inhibitor vildagliptin (31 (link)) was added. The reaction mixtures were incubated at 37°C and the OD₄₀₅ was measured at 1 min intervals for 100 min. The asparaginase assay was performed using the Asparaginase Activity assay kit (Abcam, ab107922) according to the manufacturer’s instructions. The amount of protein in BEVs was determined using the BCA protein assay kit for low concentrations (Abcam ab207002) according to manufacturer’s instructions. Protein values generally correlated with the concentration of particles determined by Zetaview (data not shown).

Total protein concentration of the collected cell lysates (n = 6) was quantified with a BCA protein assay kit for low concentrations (ab207002, Abcam, Cambridge, United Kingdom), according to the manufacturer’s protocol. Briefly, whole cell lysates with unknown protein concentration, and reference protein standard (bovine serum albumin, concentration range 0.5–40 μg/mL) were incubated with green-colored BCA Working Solution for two hours at 37 °C. The total protein concentration was then determined by measuring the color change of the sample solution from green to purple, which is proportional to the protein concentration. Absorbance was measured against λ₅₆₂ using Varioskan™ LUX multimode microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). SkanIt™ Software for Microplate Readers ver. 5.0 was used for the analysis of the data.
BDNF concentration in 1 µg of the whole cell lysate was subsequently determined using DuoSet ELISA test.

The bicinchoninic acid (BCA) assay was used to quantify the SEC fractions and serum protein content. First, 2–10 µl of each sample was added to a microplate and diluted to 10 µl (if necessary) with phosphate-buffered saline (PBS). Then, 190 µl of 49:1 bicinchoninic acid:copper sulphate solution was added to each sample and plates were incubated for 30 min at 37°C. Absorbance was read at 562 nm on a FLUOstar plate reader (BMG Labtech, Aylesbury, UK). Protein concentrations were calculated using bovine serum albumin (BSA) standards and a four-parameter logistic curve.
Conditioned cell culture media SEC fraction protein content was determined using a BCA protein assay kit for low concentrations (ab207002; Abcam, Cambridge, UK).