PMC:3537549 / 11326-13084 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":"Proliferation assay\nPBMCs were seeded immediately after isolation at a concentration of 3 × 106 cells/mL in 96-well U-bottom plates. Triplicate cultures for each cat were treated with either 4 μg/mL ODN 2216 or 2243 or an equal volume of endotoxin-free PBS. After an initial incubation of 18 h, the cells were pulsed for 24 h with 3 H-thymidine (Perkin Elmer, Schwerzenbach, Switzerland). Standard liquid scintillation protocols were used for harvesting of the cells and uptake of 3 H was assessed with the Packard Tri-Carb 1600TR liquid scintillation analyzer (Perkin Elmer). Proliferation rates were calculated as the mean counts per minute (c.p.m) of triplicate cultures. The stimulation index (SI) depicted in Figure1 was calculated as follows:Mean c.p.m.of ODN-treated culturesMean c.p.m of PBS-treated cultures\nFigure 1 ODN 2216 induce proliferation of primary feline immune cells and enhance their expression of costimulatory surface molecules. (A) The proliferation of feline PBMCs of eight adult cats belonging to groups 2 and 3 after stimulation with ODN 2216 and ODN 2243 was assessed by H3-thymidine incorporation. The dots indicate the stimulation index calculated from triplicate cultures for each cat. (B and C) Expression levels of B7.1 and MHCII surface molecules were assessed 24 h post stimulation with the indicated treatments by flow cytometry. The fluorescence of gated PBMCs of one cat selected as an example is depicted. Isotype control samples are indicated as unstained. (D-G) Mean fluorescence intensity (MFI) for B7.1 (D and E) and MHCII (F and G) of gated lymphocytes and non-lymphocyte subpopulations stimulated with the indicated treatments. Results for eight cats belonging to groups 2 and 3 are shown. *p \u003c 0.05, **p \u003c 0.01.\n","divisions":[{"label":"Title","span":{"begin":0,"end":19}},{"label":"Figure caption","span":{"begin":817,"end":1757}}],"tracks":[]}