Ex vivo T-cell proliferation assay assesses CD4⁺CD25⁺Foxp3⁺Treg cells in response to rhF8 restimulation. Splenocytes from F8^null(B6/129S) mice after single dose of R300 and rhF8 infusion were labeled with CellTrace Violet and cultured with or without rhF8 for 96 hours. Recombinant factor IX (rhF9) was used as a nonrelevant antigen control. ConA was used as a positive control for T-cell proliferation assay. Isotype IgG-treated F8^null mice were used as a control in parallel. Cells were stained for CD4, CD25, and Foxp3 and analyzed by flow cytometry for daughter cells. (A) Schematic diagram of platelet depletion and rhF8 infusion in F8^null(B6/129S). (B) Representative flow cytometry histograms of Treg cell proliferation. (C) Treg cell proliferation graph.