Goat anti human igm

Luxeptinib was provided by Aptose Biosciences. Ibrutinib (#HY-10997) was purchased from MedChem Express. Goat anti-human IgM (#109-005-043) was purchased from Jackson ImmunoResearch. Human lymphoma cell lines SU-DHL-6 (#CRL-2959), JeKo-1 (#CRL-3006), RL (#CRL-2261) and Fetal Bovine Serum (#30–2020) were obtained from ATCC. SU-DHL-6 is a human lymphoblast-like cell line, JeKo-1 is a mantle cell lymphoma cell line and RL is a human non-Hodgkin’s lymphoma B cell line. RPMI-1640 medium (#11875–093) and penicillin/streptomycin (#15070063) were purchased from Thermo Fisher Scientific. Antibodies against p-BTK (Y223) (#87141), BTK (#56044), p-PLCγ2 (#3871), p-SYK (#2710), SYK (#80460), p-BLNK (#3601), BLNK (#36438), p-CD79A (#5173), p-LYN/LCK/HCK/BLK (#70926), p-LYN (Y507) (#2731), LYN (#2796, #4576), pSrc family (#6943) and GAPDH (#2118) were purchased from Cell Signaling Technology. Phospho-BTK (Y551) (#ab40770) was purchased from Abcam. PLCγ2 (#sc-5283) and CD79A (#sc-20064) were purchased from Santa Cruz Biotechnology. All other reagents and chemicals used were of analytical grade and were obtained from Sigma-Aldrich or Thermo Fisher Scientific.

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.

Ninety-six-well polystyrene microplates were incubated with the different hemocyanins (native and deglycosylated) and with casein as a negative control, all at 10 µg/ml in PBS, through overnight incubation at 4°C. After washing with PBS, 0.05% Tween-20 (PBS-T), active solid phase sites were blocked with a 2.5% w/v casein (Winkler, Santiago, Chile) solution with a pH of 7.0, at 37°C for 2 h. Then, dilutions of 1:100 of decomplemented sera from patients and healthy donors, in blocking solution were incubated at 37°C for 1 h. Rabbit anti-human immunoglobulins (Amersham Biosciences, Buckinghamshire, UK), goat anti-human IgG (specific for Fc_γ chain, Sigma-Aldrich, St. Louis, MO, USA), or goat anti-human IgM (specific for Fc_5µ fragments, Jackson ImmunoResearch, West Grove, PA, USA), all coupled to peroxidase, were added diluted in PBS-T. The reaction was developed with ABTS and 0.1% H₂O₂, and read at 405 nm after 20 min of reaction.