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Blood, Vol. 111, Issue 7, 3813-3820, April 1, 2008
EBV LMP2A affects LMP1-mediated NF- B signaling and survival of lymphoma cells by regulating TRAF2 expression
Blood Guasparri et al.
111: 3813
Supplemental materials for: Guasparri et al
Method for establishment of IBL-1 and BCKN-1 cell lines Tissue (IBL-1) or a malignant pleural effusion (BCKN-1) samples were collected during the course of standard diagnostic procedures under sterile conditions. Mononuclear cells were isolated by Ficoll-Hypaque (Pharmacia Fine Chemicals, Piscataway, NJ) density gradient centrifugation. Cells were cryopreserved in liquid nitrogen in RPM1 solution containing 10% dimethyl sulfoxide (DMSO) and 40% fetal calf serum at −170°C. For cell line establishement, the cryopreserved cells were thawed and plated at a concentration of 2 × 106/mL on top of a layer of heterologous feeder cells. For this purpose, peripheral blood lymphocytes from a normal donor were irradiated with 50 Gy and plated at a concentration of 1.5 × l06 per well in 24-well plates 24 hours before the addition of the lymphoma cells directly to these cultures. Cells were grown in RPM1 1640 supplemented with 20% fetal bovine serum at 37°C in the presence of 5% CO2. Cell lines were passaged every 3 to 4 days after one to two cell doublings. Cell viability and evidence of cell lysis was evaluated by examination in a phase contrast microscope in the presence of Trypan blue.
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