Isolation and Characterization of i35-Breg Exosomes

Because i35-Bregs are mostly CD138⁺ plasma cells (16 (link)), splenic B cells isolated by use of MicroBeads from Miltenyi-Biotec (130-121-301) are used. For generation of i35-Breg exosomes (i35-Exosomes) the plasma cells were stimulation with anti-IgM/anti-CD40 Abs for 72 h at low density (<10⁶/ml). Analysis of aliquots for p35 and Ebi3 expression by flow cytometry, routinely showed i35-Breg enrichment (>35%) under this culture condition as previously described (7 (link)). Under this culture condition i35-Bregs or unstimulated CD19⁺ control B cells do not die as verified by Vi-Cell XR (Viability analyzer, Beckman Coulter, Indianapolis, IN). Complete media with exosome-depleted FBS was used for exosome isolation from culture supernatants of control and i35-Breg enriched cultures using Exoquick TC reagent (System Biosciences) following manufacturer's guidelines. Exosome size distribution was measured by Nanoparticle Tracking Analysis using the NanoSight system(NanoSight) and expression of exosome markers or IL-35 subunit proteins were characterized by Western blotting.