Rhodamine red conjugated anti mouse igm

Levels of circulating donor-specific antibodies (DSA; IgG and IgM) in recipient sera at the indicated time points were assessed by flow cytometry, as previously described (20 (link)). Briefly, recipient sera were incubated with C57BL/6 donor splenocytes at 37°C for 30 min, after which washed cells were incubated with fluorescein isothiocyanate (FITC)-labeled anti-mouse IgG (Abcam, ab6724, 1:100) and rhodamine red-conjugated anti-mouse IgM (Jackson ImmunoResearch Laboratories, 115-297-020, 1:100) at 4°C for 1 h. Cells were analyzed by FACScan (Becton–Dickinson, Lincoln Park, NJ, United States) flow cytometry with results expressed as mean fluorescence intensity to reflect individual serum DSA levels.

Circulating donor-specific IgG and IgM antibodies were assessed in recipient sera by flow cytometry. In short, recipient sera were incubated with C3H donor splenocytes at 37°C for 30 min, and washed cells were then incubated with FITC-labeled anti-mouse IgG (Abcam, Cambridge, England) and rhodamine red-conjugated anti-mouse IgM (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) at 4°C for 1 h. Cells were analyzed by flow cytometry with results expressed as mean fluorescence intensity to reflect individual serum antidonor antibody levels.