Journal:

Article Title: Interleukin-12 Is Essential for a Protective Th1 Response in Mice Infected with Cryptococcus neoformans

doi:

Figure Lengend Snippet: Net cytokine secretion of total spleen cells from mice infected with C. neoformans. Spleens were taken from three mice per group 21 days after infection with 104 CFU of C. neoformans. Spleen cells were depleted of CD4+ or CD8+ T cells by using specific antibodies and magnetic beads. As assessed by subsequent fluorescence-activated cell sorter analysis, depletion of CD4+ or CD8+ T cells was complete (100%). The graph shows cytokine secretion after specific stimulation with heat-killed cryptococci with background values (medium controls) subtracted. The levels of IFN-γ and IL-4 in splenocyte cultures incubated with medium for 48 h were less than 5 and 10%, respectively. Polyclonal stimulation with anti-CD3 was similar between different groups (not shown). Levels of IFN-γ and IL-4 were quantified in 48-h culture supernatants by an enzyme-linked immunosorbent assay. Since the concentration of cells was kept constant at 2.5 × 106 cells/ml in total and depleted samples, bars representing the CD8+-depleted samples reflect CD4+-enriched cultures with elevated cytokine production. This figure demonstrates the results of one representative experiment of nine similar experiments with cytokine analyses of lymphocytes cultured ex vivo.

Article Snippet: In order to prevent autoconsumption of IL-4, neutralizing rat anti-murine IL-4 receptor antibody (Genzyme, Cambridge, Mass.) was added to the cultures (5 μg/ml).

Techniques: Infection, Magnetic Beads, Fluorescence, Incubation, Enzyme-linked Immunosorbent Assay, Concentration Assay, Cell Culture, Ex Vivo