Magellan software v7

The immunomodulatory properties of Exo MSC and Exo MSC-NTF were evaluated in vitro by examining inhibition of cytokine secretion by peripheral blood mononuclear cells (PBMCs) in response to activation with phytohemagglutinin (PHA). PBMCs (5 × 10⁵) were stimulated with 10 μg/ml PHA and incubated with Exo MSC or Exo MSC-NTF (2 × 10⁹ particles) for 4 days in culture. IFNγ and TNFα were measured in the culture supernatant using a commercial ELISA (DuoSet ELISA, R&D Systems, Minneapolis, MN) that was read at 450 nm with Sunrise plate reader and analyzed by the Magellan Software V7.2 (Tecan, Switzerland).

To measure the content of specific proteins in sEVs, 1 ml of sEV enriched fractions was precipitated using ExoQuick-CG (SBI, USA). EV pellets were lysed using M-PER Mammalian Protein Extraction Reagent (ThermoFischer, USA), supplemented with 1:200 Protease Inhibitor Cocktail Set III, EDTA-Free (Calbiochem). Following 10-min incubation in room temp, the lysates were frozen and thawed twice to ensure complete lysis. Lysates’ protein concentrations were measured using BCA kit (ThermoFischer, USA) and concentrations of 60–75 μg/ml were used for ELISA assays. Amphiregulin (AREG) and LIF concentrations were measured using Quantikine kits (R&D Systems, Minneapolis, MN; Cat# DAR001, DLF00B). HGF and TSG-6 concentration were measured with ELISA kits from RayBiotech, USA (Cat# ELH-HGF-CL-1, ELH-TSG6-1). Signals were quantified using Sunrise plate reader and the Magellan Software V7.2 (Tecan, Switzerland).