Figure 6

Spleen cell growth and cytokines secretion assays. - A: Spleen cell growth determined by MTS. Spleen cells from normal Balb/c mouse (2 × 10⁵ cells per well) were seeded in triplicate in a 96-well plate which was coated with anti-mouse CD3 mAb. Then, the cells were incubated with the purified recombinant B7-H4 protein, 3E8 mAb, or their admixture which was premixed for 30min. Normal mouse IgM was used as an antibody control. After incubating the wells for 72 hours at 37°C the wells were added with MTS. After incubating the wells for 4 hours at 37 OQ the OD of each well was measured using a Model 680 microplate reader at a wavelength of 490nm. The numbers of living cells were presented by OD490. B: Cytokines secretion determined by ELISA. Spleen cells were treated as above. The Cell culture supernatant was collected respectively after incubating the wells for 48 or 72 hours at 37°C The expression of IL-2, IL-4, IL-10 in 48h supernatant, and IFN-γ in 72h supernatant were determined with mouse cytokine ELISA kit. The expression levels of cytokines were presented by OD450. *: vs Lane 33 and Lane 5, P <0.05.