In ux cell sorter

Amelanotic tyrosinase-knockout B16-F10 melanoma cells were kindly provided by Dr Shweta Tikoo from the Centenary Institute, Sydney, Australia, and maintained in Advanced Dulbecco's modi ed Eagle's medium and 5% fetal bovine serum (Thermo Fisher Scienti c, Waltham, USA). They were stably transfected with CEFLP-tdTomato-H2B eukaryotic expression plasmids using GenePORTER®3000 (Genlantis, San Diego, CA, USA). Transfected cells were selected with puromycin (10 µg/mL) and tdTomato-expressing cells were puri ed by ow cytometry on a BD In ux Cell Sorter.

Mediastinal lymph nodes and lungs were harvested at indicated days of infections and processed to generate single cell suspension as previously described 12, 55 . A list of all antibodies used for ow cytometery in this study is in Supplemental Table 2. In general, cells were rst stained with surface markers at 30 min room temperature (RT) and an additional 30 min on ice. Cells were then xed and permeabilized with Foxp3 x/perm (Tonbo) before being stained for intracellular markers for 30 min on ice. For OTII TCR speci c tetramer staining, cells were incubated with I-A(b) AAHAEINEA tetramers in 50µl for 1hr in 37ºC incubator before addition of other surface markers with additional 30 min at RT before xation as described above. For Annexin V staining, cells were stained for surface markers and washed before staining for Annexin V for 20 min at RT. Cells were washed and quickly analyzed in a cytometer. All ow cytometry samples were acquired using LSRII cytometry (BD) with FACSDiva software. All cell sorting were performed in BD In ux cell sorter. Data were analyzed using FSC Express (DeNovo software).

Amelanotic tyrosinase-knockout B16-F10 melanoma cells were kindly provided by Dr Shweta Tikoo from the Centenary Institute, Sydney, Australia, and maintained in Advanced Dulbecco's modi ed Eagle's medium and 5% fetal bovine serum (Thermo Fisher Scienti c, Waltham, USA). They were stably transfected with CEFLP-tdTomato-H2B eukaryotic expression plasmids using GenePORTER®3000
(Genlantis, San Diego, CA, USA). Transfected cells were selected with puromycin (10 µg/mL) and tdTomato-expressing cells were puri ed by ow cytometry on a BD In ux Cell Sorter.