Human b cell isolation kit

PBMC’s were isolated from leukocyte reduction cone obtained from Degowin Blood Center at the University of Iowa Hospital and Clinics. Donors are anonymous and have provided written informed consent to allow their cells that are normally discarded to be used in research studies. The recruitment protocol and written informed consent document were approved by the Institutional Review Board for the University of Iowa. All samples were provided to investigators de-identified. Therefore, further IRB approval for the use of the cells by the investigators was not needed based on Federal Regulation 46.101.B4. All experiments were performed in accordance with approved guidelines. PBMC’s were isolated via density gradient using Lymphoprep (StemCell Technologies 07801). B cells were isolated from PBMCs using a negative selection Human B Cell Isolation kit (StemCell Technologies 17954) following the provided protocol. B cells were rested at 37 °C in Complete RPMI 1640 media until used and used within 5 h of negative selection.

Whole blood from SLE patients and healthy controls was collected into sodium-heparin tubes (BD Biosciences, San Jose, CA, USA) and diluted 1:1 with PBS prior to isolation of peripheral blood mononuclear cells (PBMCs). PBMCs were purified by density gradient using Ficoll-Paque (GE Healthcare Life Science, Marlborough, MA, USA) in Leucosep tubes (Greiner Bio One, Monroe, LA, USA) per protocols provided by the manufacturer. Blood plasma was collected after centrifugation of blood. The PBMC layer was extracted and washed twice with PBS prior to cryopreservation in Recovery Cell Culture Freezing Medium (Gibco, Grand Island, NY, USA) at 10⁷ PBMCs per ml. PBMC vials were frozen at -80°C overnight prior to long-term storage in liquid nitrogen. Plasma was aliquoted and stored at -80°C until further use. For in vitro experiments, B cells were isolated from PBMCs derived from healthy donor whole blood buffy coats available from the Stanford Blood Center. B cells were subsequently isolated using the Human B Cell Isolation Kit (Stemcell Technologies, Vancouver, BC, Canada) according to manufacturer’s protocol. Purity and viability was >97% as assessed by flow cytometry.

B cell lines (BCL) were generated using PBMC and immortalized with Epstein-Barr virus (EBV). Frozen PBMC were thawed and incubated with 2.5 mL of supernatant from B95-8 cell lines for 2 h in a 37°C water bath. A total of 1 μg/mL of cyclosporine in 5 mL of R10 was then added to the cell suspension and incubated in a T25 flask for 3 weeks at 37°C. B cells that were difficult to immortalize in this manner were first purified using a human B cell isolation kit (STEMCELL) and then incubated with B95-8 supernatant.