Fitc anti il 2 and pe anti ifnγ

Splenocytes from immunized mice were also assayed by ICS for interleukin 2 (IL-2) and IFN-γ. The cell suspensions, obtained as described above, were suspended to a final concentration of approximately 10⁷ cells/ml. All groups of harvested spleen cells (1 X 10⁶ cells / tube) were stimulated for 18 h with (1) medium alone, (2) whole heat-inactivated YF 17D virus, (3) pool of peptides described above or (4) concanavalin A (2 μg/ml; Sigma, St. Louis, MO) in the presence of 1 μg of anti-CD28 and anti-CD49d monoclonal antibodies/ml (BD PharMingen, San Diego, CA). Five hours before the end of the incubation, 10 μg/ml of brefeldin A (Sigma, St. Louis, MO) was added to each tube. Cells were then washed (PBS 10% FCS) and stained for surface antigens with PerCP anti-CD8 and Alexa 647 anti-CD4 mouse monoclonal antibodies (BD-Biosciences PharMingen, San Diego, CA). The cells were washed again, fixed with paraformaldehyde 2%, permeabilized (PBS 10% FCS 0,1% Saponin) and stained with monoclonal antibodies FITC anti-IL-2 and PE anti-IFN-γ (BD-Biosciences PharMingen, San Diego, CA) followed by multiparametric flow cytometry on FACScalibur flow cytometer using CellQuest software (BD Biosciences, San Jose, CA). The data were analyzed with FlowJo 7.6.5 for Windows software (Tree Star, Ashland, OR).