FIGURE 7.

Effect of recombinant human Hsp72^bv on splenocyte functions. Mouse splenocytes (10⁶ cells) were treated with BSA (100 μg) or recombinant Hsp72^bv (100 or 200 μg) for 96 h in a 37 °C incubator. A, supernatant was recovered, and IL-4, tumor necrosis factor-α, IL-12p70, or IFN-γ was measured using cytometric bead assay according to the manufacturer's instructions (BD Biosciences) on a BD FACSAria flow cytometer. Raw data were analyzed using FCAP array software. Data are mean fluorescence intensity ± S.D. and are the sum of three independently performed experiments. *, p < 0.05 versus control (BSA). B, splenocytes were collected and stained with anti-mouse CD11-phycoerythrin, CD4-fluorescein isothiocyanate, and CD8-phycoerythrin and analyzed using a BD FACSAria flow cytometer. Individual cells were gated on the basis of forward and orthogonal scatter. The photomultiplier for fluorescein isothiocyanate (FL1-height) or PE (FL2-height) was set on a logarithmic scale. Cell debris was excluded by raising the forward scatter-height photomultiplier tube threshold. The flow rate was adjusted to <200 cells/second, and at least 30,000 cells were analyzed for each sample. Data are the sum of four independently performed experiments. *, p < 0.05 versus control (BSA).