Anti h 2kq fitc

Chimerism levels were examined at the age of 1 month old. Peripheral blood was sampled via tail veins and depleted of red cells using ACK lysing buffer. Cells were first incubated with anti-mouse FcγII/FcγIII antibody (BioLegend) and then stained with anti-H-2K^q FITC (BioLegend) and anti-H-2K^b PE (BioLegend). Chimerism levels were determined by flow cytometry after gating out dead cells by high propidium iodide staining. A negative control consisted of anti-H-2K^q FITC and mouse IgG2a PE (BioLegend) to define background staining. Recipients with first month peripheral chimerism of >3% were collected to further validate tolerance by skin transplantation because they could be consistently rendered tolerant to donor skin [11 (link)]. As for the quantification of donor leukocyte's MHC-I expression, engrafted donor leukocytes were gated, and the fluorescence intensities (FIs, including mean, geographic mean, and median) of anti-H-2K^b PE were measured. Wild-type C57BL/6 mice were used as controls.