Rpmi 1640 glutamax

Neuroblastoma cancer cells, namely SK-N-SH (ATCC, ref. HTB-11) and IMR-32 (ATCC, ref. CCL-127) cells, were purchased from the American Type Culture Collection and routinely maintained in standard RPMI 1640 (L-glutamine +) culture medium (Fisher Gibco^™ RPMI-1640 Glutamax^™) supplemented with 10% fetal bovine serum (Lonza) and 1% penicillin-streptomycin 5000 U/mL (Fisher Gibco^™ Pen Strep) at 37 °C and 5% CO₂. Cell cultures between 3 and 17 passages from defrosting were used for MTT assays.

B cells were stimulated in vitro with CpG 2006 oligonucleotide (CpG) (TIB MolBiol Synthese Labor GmbH, Berlin, Germany). The cells were resuspended in RPMI 1640 Glutamax supplemented with 10% FCS (Lonza, Köln, Germany), 5% penicillin/streptomycin, and 0.05 mM 2-mercaptoethanol (Gibco® Life Technologies GmbH, Darmstadt, Germany). B cells, 10⁵, were seeded and stimulated with 2.5 μg/mL CpG for 48 h at 37°C and 5% CO₂. After 2 days of culture, the supernatants were harvested and frozen at −70°C prior to analysis.
To analyze the IL-10 production by B cells, PBMCs (10⁶/well) were cultured with CpG 2006 in vitro as described [26 (link),27 (link)]. Intracellular staining of IL-10 and Ki67 was performed on PBMCs after 2 days of culture. PBMCs were re-stimulated for 4 h with 10 ng/mL PMA and 1 μM ionomycin including 2 μg/mL brefeldin A for the last 2 h (all from Sigma Munich, Germany) prior to intracellular staining. Unstimulated cells served as controls.

Mice were sacrificed post-infection with pentobarbital (CEVA santé animale; Libourne, France) diluted 1∶5 with saline. Lungs were flushed with 500 µl of saline to obtain BAL fluids. Cells were isolated from the peritoneal cavity by washing with ice-cold RPMI 1640 (Glutamax; Lonza, Basel, Switzerland). Blood was taken by intra-cardiac puncture with a 1 ml syringe containing 100 µl of heparin (100 U.I/ml) (Sanofi-Synthelabo, Le Plessis Robinson, France). Blood, BAL and PL fluids were plated at various dilutions on TSA Petri dishes for colony forming unit (CFU) counting, the remaining volume was centrifuged at 300 g for 10 min at 4°C. The leukocyte pellet from BAL and PL fluids was analyzed by flow cytometry. Supernatants and plasma were filtered (0.22 µm) using 160 Spin X columns (CoStar, Corning Lifesciences, Lowell, MA, USA) and kept at −20°C for cytokine dosage.