Ficoll gradient centrifugation

Peripheral blood mononuclear cells (PBMC) were isolated from buffy coat by Ficoll gradient centrifugation (GE healthcare) and washed three times in PBS. T cells were negatively selected with magnetic-assisted cell sorting (MACS) from PBMC with the use of CD8⁺ T cell isolation kit or Pan T cell isolation kit (Miltenyi Biotec). Primary human T cells were cultured in RPMI 1640 medium (Gibco), supplemented with 10% heat inactivated FBS (Gibco), and 1% Penicillin and Streptomycin (Gibco). Monocytes were positively selected with MACS by CD14 Microbeads (Miltenyi Biotec) and were cultured in RPMI 1640 medium (Gibco), supplemented with 10% heat inactivated FBS (Gibco), and 1% Penicillin and Streptomycin mixture (Gibco).

Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll gradient centrifugation (GE Healthcare, Little Chalfont, UK) from whole blood. PBMCs were cultured at a density of 1 × 10⁶ cells/mL in 24-well plates (Corning, New York City, NY, USA) in RPMI 1640 with 10% of Fetal Bovine Serum (FBS), in the presence or absence of Resiquimod (R848) (Invivogen, San Diego, CA, USA) (100 ng/mL) for 6 hours at 37 °C. Then, cells were collected and stained with specific antibodies discriminating viable pDCs and mDCs and evaluating their expression of activation markers. After the isolation, the cells were stained with specific antibodies enabling to discriminate mDCs and pDCs.

Complete blood counts (CBC), analysis of lymphocyte subsets and serum immunoglobulin values were determined according to routine and accredited laboratory methods (http://www.huslab.fi). Mononuclear cells (MNCs) were isolated from peripheral blood by Ficoll gradient centrifugation (GE healthcare, Buckinghamshire, UK).

Peripheral blood specimens were collected from PTCL patients at the University of Maryland Greenebaum Comprehensive Cancer Center with the approval of the University of Maryland, Baltimore Institutional Review Board (UMB IRB). Written consent was obtained from all patients using a UMB IRB approved consent procedure. Peripheral blood mononuclear cells were isolated from each specimen by Ficoll gradient centrifugation according to the manufacturer's protocol (GE Healthcare). Cells were cultured for up to 48h with IL-2 stimulation.

Analysis of samples from the healthy volunteer and the patient were approved by the Human Research Ethical Committees of Ehime University (No. 1712006) and Shinshu University with the patient-supplying written informed consent (No. 476). All experiments were performed in accordance with the relevant guidelines and regulations for human samples.
All normal human PBMCs used in the study were separated from a healthy male volunteer. Human PBMCs from the healthy control and the patient with MWS were separated by Ficoll-gradient centrifugation, according to the manufacturer's instructions (GE Healthcare Biosciences AB, Piscataway, NJ, USA). The patient with MWS is a 21-year-old Japanese woman diagnosed with a CAPS/MWS at the age of 7 years; she harbors an arginine-to-tryptophan mutation at position 260 (R260W) of NLRP3^{25 (link)}. She was being treated with injections of canakinumab (150 mg every 2 months); therefore, her PBMCs were separated just before injection to avoid the effects of medication.