PMC:3537549 / 9557-11324 JSONTXT

{"project":"2_test","denotations":[{"id":"22906110-9765447-143530971","span":{"begin":219,"end":221},"obj":"9765447"},{"id":"22906110-20133174-143530972","span":{"begin":390,"end":392},"obj":"20133174"}],"text":"Feline PBMC isolation, cell lines, ODNs, cell culture and cell viability assay\nFeline PBMCs were isolated from EDTA-anticoagulated whole blood by Ficoll-Hypaque density gradient centrifugation using a standard protocol[56]. Purified cells were counted prior to their utilization in the different experiments using the Sysmex XT 2000iV (Sysmex, Norderstedt, Germany) as described previously[57], and cultured in RPMI 1640 with Glutamax I (Gibco®, Invitrogen, Basel, Switzerland). Adherent CRFK (ATCC no. CCL-94) and FEA cells were maintained in RPMI 1640 with Glutamax I, while adherent fcwf-4 cells (ATCC no. CRL-2787) were cultured in EMEM (ATCC 30–2003). All media were supplemented with 10% heat-inactivated fetal calf serum (Bioconcept, Allschwil, Switzerland), 100 U/mL penicillin and 100 mg/mL streptomycin (Gibco®, Invitrogen).\nODN 2216 and ODN 2243 were obtained from Alexis biochemicals, Enzo Life Sciences AG, Switzerland for in vitro studies and ODN 2216 was synthesized by Microsynth AG, Balgach, Switzerland for the in vivo experiment. Recombinant feline IFNα (rfeIFNα) was obtained from PBL Biomedical, Piscataway, New Jersey, USA. All molecules were solubilized in endotoxin-free PBS. ODN 2243 consists of the same sequence as ODN 2216, with CpG motifs inversed to GpC. For in vitro experiments, both ODNs and rfeIFNα were diluted in RPMI 1640 with Glutamax I supplemented as described above.\nViability of stimulated cells was compared using the trypan blue exclusion test. Briefly, after stimulation for 24 h with increasing concentrations of ODN 2216, ODN 2243 or equivalent volumes of PBS as control, cells were stained with a 0.4% trypan blue solution (Dr Bender and Dr Hobein AG, Zurich, Switzerland) and percentages of viable cells were compared."}