Bioplex human 27 plex

Concentrations of 27 human cytokines were simultaneously evaluated using a commercially-available multiplex bead-based sandwich immunoassay kit (BioPlex human 27-plex, catalogue number M50-0KCAF0Y, BioRad Laboratories, Hercules, CA, USA). Cytokines were collected in two fractions at the same passage from each of the cell lines in biological triplicate—secreted from the cells into the media (referred to as conditioned media or CM) and associated with the EVs. Isolated EVs were lysed, to release encapsulated cytokines, using a probe sonicator (Sonics & Materials, Inc., CT, USA) in PBS. All samples were centrifuged at 20,000× g for 10 min to remove any cellular debris. Assays were performed according to the manufacturer’s instructions, as previously described [62 (link)]. Images of results were generated in MS Excel, DanteR (Version 1.0.0.10, Richland, WA, USA) and GraphPad Prism (Version 8.2.1, San Diego, CA, USA). For the analysis and comparison of the EV samples, statistical analyses were performed using GraphPad Prism. The data were tested with a one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test. Data are represented as means ± standard deviation (SD). A p value of <0.05 was considered statistically significant.