PMC:3537549 / 13085-14762 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/3537549","sourcedb":"PMC","sourceid":"3537549","source_url":"https://www.ncbi.nlm.nih.gov/pmc/3537549","text":"Flow cytometry\nPBMCs were treated at a density of 3 × 106 cells/mL with 4 μg/mL ODN 2216 or ODN 2243 or an equivalent volume of endotoxin-free PBS and cultured for 24 h in a 12-well format. During collection of the cells, the adherent cell fraction was removed with 0.05% trypsin-EDTA (Gibco®, Invitrogen). Harvested cells were divided into 3 fractions labeled separately with either anti-feline B7.1 mouse monoclonal IgG (kindly provided by Prof Mary Tompkins, Flow Cytometry and Cell Sorting Laboratory, NC State College of Veterinary Medicine, USA), anti-feline MHCII mouse monoclonal IgG1 (Department of Pathology, Microbiology and Immunology, University of California, Davis, USA) or fluoresceinisothiocyanate (FITC)-conjugated mouse IgG1 as isotype control (BD Bioscience, Allschwil, Swizerland). The fractions were subsequently stained with R-Phycoerythrin (RPE)-conjugated goat anti-mouse IgG1 (BioConcept, Allschwil, Swizerland). Fluorescence data was obtained using the FACSCalibur® instrument (Becton Dickinson, Allschwil, Switzerland) and the CellQuestPro™ software. Gates representing lymphocyte and non-lymphocyte populations were set on the basis of forward versus side scatter, and a total of 50 000 events were acquired in the non-lymphocyte gate. Data was analyzed with the FlowJo software (Tree Star, Olten, Switzerland), whereby an additional gate was set comprising both lymphocyte and non-lymphocyte populations (PBMC gate). MHCII and B7.1 expression levels were determined as mean of fluorescence intensity for each gated cell population. Identical gates were set for all cats in such a way that they comprise the desired cell populations of each animal.","divisions":[{"label":"Title","span":{"begin":0,"end":14}}],"tracks":[]}