Thy1.1+ pmel T cells were harvested from the spleens of Pmel transgenic mice and negatively selected for CD8 with a magnetic bead enrichment method (STEMCELL Technologies). Cells were labeled with 5 μM of CSFE (Thermo Fisher) and washed 3 times with PBS and injected i.v. at 1 × 105 cells per mouse.
For adoptive transfer of NK cells and macrophages, endogenous cells were depleted with neutralizing antibodies against NK cells and macrophages in B16F10-bearing Fcgr3-KO or WT mice 1 day before the treatment. Syngeneic WT CD45.1+ or Ifng-KO NK cells and macrophages were harvested and selected using a magnetic bead enrichment method (Miltenyi Biotec) and Ifng-KO cells were labeled with 5 μM CellTrace Violet or CFSE (Thermo Fisher). NK cells and macrophages were then i.v. injected at 1 × 106 cells (Tai et al., 2013 ) and 3–4 × 106 cells (Hagemann et al., 2008 (link)) per mouse, respectively.
For adoptive transfer of NK cells and macrophages, endogenous cells were depleted with neutralizing antibodies against NK cells and macrophages in B16F10-bearing Fcgr3-KO or WT mice 1 day before the treatment. Syngeneic WT CD45.1+ or Ifng-KO NK cells and macrophages were harvested and selected using a magnetic bead enrichment method (Miltenyi Biotec) and Ifng-KO cells were labeled with 5 μM CellTrace Violet or CFSE (Thermo Fisher). NK cells and macrophages were then i.v. injected at 1 × 106 cells (Tai et al., 2013 ) and 3–4 × 106 cells (Hagemann et al., 2008 (link)) per mouse, respectively.