Flow cytometric analysis of the kinetics of immunoglobulin subclasses in presensitized mice. Donor splenocytes (0.5 × 10⁶) from naive B10.BR or BALB/c mice were incubated with 5 μL sera from sensitized B10 or B6 recipients and age-matched naive controls for 30 minutes, then incubated with FITC-conjugated goat anti-mouse immunoglobulin and goat anti-mouse IgM, IgG, IgG1, IgG2a, or IgG2b, followed by incubation with PE-conjugated anti-mouse CD4 plus CD8. Levels of circulating alloantibodies were determined by FACSCalibur gating on the CD4⁺ and CD8⁺ T cells and were reported as MFI. (A) Antidonor Ab titers were detected with staining of goat anti-mouse immunoglobulin in B6 or B10 mice before placement of the donor skin graft (week 0) or at 4 weeks after skin grafting. (B) The kinetics of IgM and IgG were analyzed in presensitized mice up to 13 weeks. (C) Isotypes of IgG (IgG2a, IgG2b, IgG1) were tested with sera collected before (week 0 [w0]) and 4 to 6 weeks after skin grafting. Results from sera tested at week 5 are presented. IgM was tested with sera collected before and 2 weeks (w2) after skin grafting. Data are representative of 1 of 4 separate experiments. (D) Sera were incubated with donor BMCs, and the expression of IgG on KSL cells from sensitized mice at 4 to 12 weeks was compared with the control collected before graft placement. Data are representative of 1 of 3 separate experiments. Error bars indicate SD.