Oci ly1

Non-GCB type DLBCL cell-line NU-DUL and GCB type DLBCL cell-lines OCI Ly1 and OCI Ly18 were purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Germany). Non-GCB type DLBCL cell-lines OCI Ly3, OCI Ly10 and GCB type DLBCL cell-line OCI Ly19 were a kind gift of Dr. F. Bertoni, Bellinzona, Switzerland. NU-DUL were maintained in RPMI 1640 medium supplemented with 15% fetal bovine serum (FCS) + 2-Mercaptoethanol (50mM; Invitrogen); OCI Ly10, OCI Ly3 and OCI Ly1 in IMDM 20% FCS + 2-Mercaptoethanol (50mM); OCI Ly18 in RPMI 10% FCS + 2-Mercaptoethanol (50mM) and OCI Ly19 in RPMI 10% + 2-Mercaptoethanol (50mM) + MEM NEAA + sodium pyruvate. BL Raji cell-line was purchased from ATCC and cultured in RPMI 20% FCS. Testing for Mycoplasma infection was carried at a monthly basis. Peripheral blood mononuclear cells (PBMCs) and B lymphocytes from healthy donors were obtained from peripheral blood as per standard Ficoll Paque^® protocol; B lymphocytes were purified using CD19-coated magnetic MicroBeads according to the manufacturer's protocol (Miltenyi Biotech).

The 16 DLBCL cell lines (DOHH2, HT, OCI-LY19, DB, OCI-LY1, SUDHL-4, SUDHL-5, SUDHL-10, NUDHL-1, OCI-LY7, WSU-DLCL2, SUDHL-6, NUDUL-1, U2932, OCI-LY3, and RI-1) were purchased from the American Type Culture Collection or from DSMZ (Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Germany). They were grown in RPMI-1640 Glutamax medium (Gibco, Invitrogen, Cergy Pontoise, France), supplemented with 10% fetal bovine serum (FBS) (PAA laboratory GmbH, Pasching, Austria) (U2932, SUDHL-4, HT, DOHH2, SUDHL-10, RI-1, and WSU-DLCL2 cells), 20% FBS (OCI-LY3, DB, SUDHL-5, NUDHL-1, and SUDHL-6 cells), or 15% FBS (NUDUL-1 cells). OCI-LY1, and OCI-LY7 cells were cultured in IMDM Glutamax (Gibco, Invitrogen, Cergy Pontoise, France), supplemented with 20% FBS, and OCI-LY19 cells in MEM alpha modified Glutamax (Gibco, Invitrogen, Cergy Pontoise, France) with 20% FBS. Cultures were maintained at 37 °C in a humidified atmosphere with 5% CO₂. Contamination by Mycoplasma species was regularly monitored.

Splenic B cells were cultured (10⁶ cells/ml) in RPMI (Euroclone) + 10% FCS (Euroclone) and 2-mercaptoethanol (50 mM; Life Technologies) for 72h with 20 μg/ml LPS (Sigma) ± 10 ng/ml IL-4 (Sigma) or for 48h with 10 μg/ml anti-CD40 (BD Pharmingen) ± 10 ng/ml IL-4 (Sigma). To perform fast stimulation 5x10⁶ B cells were resuspended in 400ul PBS without FCS and treated for 5 and 10 minutes with 10μg/ml anti-CD40 (BD Pharmingen) ± 10 ng/ml IL-4 or for 1, 5, 10 minutes with 10 μg/ml anti-Mouse IgM (Jackson Immuno Research); the incubation was performed in water bath at 37°C followed by the addiction of ice-cold PBS to stop the stimulation; cells were immediately pelleted and lysed. DLBCL cell lines OCI-Ly1, OCI-Ly18 and Pfeiffer were purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Germany). OCI-Ly1 were maintained in IMDM (Invitrogen) + 20% FBS + 2-mercaptoethanol (50 mM); OCI-Ly18 in RPMI + 10% FBS + 2-mercaptoethanol (50 mM); Pfeiffer in RPMI + 10% FBS + Sodium Pyruvate 1 mM. The cells were maintained in an incubator at 37°C in a modified atmosphere with 5% CO2. Testing for Mycoplasma infection was carried out on a monthly basis. All procedures of handling were carried out under a sterile hood.