Purified CD43− quiescent B cells were labeled with CFSE (Molecular Probes Invitrogen, Karlsruhe, Germany) as described previously for PBMC proliferation for 7 min at 37°C. After labeling, B cells were resuspended in IMDM with 10% v/v heat inactivated FBS supplemented with a cocktail to mimic T cell activation, composed of 1,000 UI/mL IL-2 (Proleukin, Novartis, Prometheus laboratories Inc., San Diego, CA, USA), 10 μg/mL Goat anti-human IgM (Jackson Immunoresearch, Cambridgeshire, UK), and 5 μg/mL soluble recombinant human CD40L (Biolegend, San Diego, CA, USA). One hundred microliter per well of the previously described cell suspension were added to the previously seeded pMSCs at a 1:5 MSC:B cell ratio and co-cultured for 7 days.
B cell proliferation was characterized by flow cytometry (FACS Canto II) and analyzed with FlowJo V10.07 (BD Biosciences). Samples were collected by careful aspiration and centrifuged for 5 min at 690 g, and supernatants were stored at −80°C for IgG quantification. Cells were stained using the following flow cytometry antibodies: CD19-BV512 (clone HIB19), CD27 PE-Cy7 (clone 0323), CD38-PE (clone HB7), Viaprobe (BD Biosciences, San Jose, CA, USA). A total of N = 3 different B cell donors co-cultured with one pMSC donor in duplicates or triplicates were analyzed.
B cell proliferation was characterized by flow cytometry (FACS Canto II) and analyzed with FlowJo V10.07 (BD Biosciences). Samples were collected by careful aspiration and centrifuged for 5 min at 690 g, and supernatants were stored at −80°C for IgG quantification. Cells were stained using the following flow cytometry antibodies: CD19-BV512 (clone HIB19), CD27 PE-Cy7 (clone 0323), CD38-PE (clone HB7), Viaprobe (BD Biosciences, San Jose, CA, USA). A total of N = 3 different B cell donors co-cultured with one pMSC donor in duplicates or triplicates were analyzed.