Fitc anti mouse cd8a 53 6

Peripheral blood and spleens were used to detect the frequency of H-2K^b alloantigen-reactive CD8⁺ T cells. On day 15 after skin transplantation, spleens and peripheral blood were collected from recipient bm1 and processed to cell suspensions. Firstly, anti-mouse CD16/CD32 (2.4G2, BD Biosciences) was used to block the cells (1 μg/8 × 10⁵ cells) for 30 min. Then, the cells were then incubated for 1 h with the mixture of APC-anti-mouse IgG1 and peptide-unloaded H-2K^b-Ig dimer (BD Biosciences) at 4 °C for 1 h. The cells were washed twice and incubated again for 30 min with PE-anti-mouse CD3e (145-2C11, eBiosciences) and FITC-anti-mouse CD8a (53-6.7, eBiosciences). Then, cells were washed with sterile PBS and acquired on a FACS Calibur flow cytometer (BD Biosciences). The data were analyzed by using the flowJo software (Tree Star, Ashland, OR).

Frequencies of B cells, NK cells, CD3⁺ T cells, CD4⁺ T cells and CD8⁺ T cells were enumerated in spleen cells from the recipient bm1 mice treated by killer NPs, blank NPs or PBS on day 15 after skin graft transplantation. Splenocytes (2 × 10⁵) were stained with PE-anti-mouse CD4 (GK1.5, eBiosciences), APC-anti-mouse CD3e (145-2C11, eBiosciences), FITC-anti-mouse CD19 (MB19-1, Biolegend), FITC-anti-mouse NK1.1 (PK136, Biolegend) and FITC-anti-mouse CD8a (53-6.7, eBiosciences), respectively, for 30 min at 4 °C. The cells were then washed with PBS and analyzed by using flow cytometry and flowJo software. In parallel, peripheral blood was collected from orbital venous of recipient bm1 mice on days 15, 30 and 45 post-transplantation. Routine blood tests were performed by automated hematology analyzer (Sysmex XE-2100, Kobe, Japan).