Anti h 2kb pe

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.

Single-cell suspensions of the aGVHD target organs, including spleen, liver, lung, and intestine, were prepared as previously described (16 (link)). Antibodies used for flow cytometry staining including anti-CD69-PerCP/Cy5.5, anti-CD3-PE/CF594, anti-CD8-Pacific Blue, anti-CD4-APC/CY7, anti-CD25-PE, anti-FoxP3-APC, anti-CD4-PE/CF594, were purchased from BD Biosciences (Franklin lakes, NJ). Anti-IFN-γ-APC, anti-TNF-α-PE/CY7, anti-IL-17A-PerCP/Cy5.5, anti-H-2k^b-PE, anti-H-2k^d-FITC, anti-CD45.2-APC, anti-CD45.1-APC/CY7 were purchased from Biolegend (San Diego, CA). For hepatocytes isolation, single liver cells were resuspended in 50% Percoll solution, centrifuged at 2,000 rpm for 20 min. The purified anti-mouse CD16/32 antibody was purchased from eBioscience (San Diego, CA). Intercellular staining and Treg detection were performed by using CytoFix/CytoPerm buffer (BD Biosciences, San Diego, CA) and FoxP3 staining Kit (eBioscience, San Diego, CA), respectively according to the manufacturer's instructions. Samples were detected on a NovoCyte Flow Cytometer (ACEA Biosciences, San Diego, CA) and data were analyzed by using Flowjo software (Flowjo, Ashland, OR).