Expression profiling of tumor-infiltrating CD4+ FOXP3- conventional T cells from tumors of wildtype and Foxp3-Cre x CK2b-fl/fl mice

Analysis of tumor-infiltrating CD4+ FOXP3- conventional T cells from tumors of Rosa26-RFP x Csnk2bfl/fl mice (wildtype) and Rosa26-RFP x Csnk2bfl/fl x Foxp3-Cre mice. B16.F10 melanoma cells (2x10e5 in 100 µl PBS) were injected subcutaneously (s.c.) into the right flank of 10-16-week-old syngeneic male and female Rosa26-RFP x Csnk2bfl/fl and Rosa26-RFP x Csnk2bfl/fl x Foxp3-Cre mice. 21 days post-injection the mice were euthanized via CO2 inhalation. By using a scalpel, the tumors were carefully separated from the inside of the skin. Excised tumors were cut into small pieces (2-4mm), transferred into gentleMACS™ C tubes (Miltenyi Biotec; 130-093-237) and digested with the Tumor Dissocation Kit, mouse (Miltenyi Biotec; 130-096-730) according to the manufacturer's instructions using the gentleMACS™ Octo Dissociator with Heaters (Miltenyi Biotec; 130-096-427). For subsequent cell sorting, the cell suspensions were strained two subsequent times using a 70 µm and then a 40 µm cell strainer and resuspended in FACS buffer (PBS + 0.5% BSA + 0.01% sodium azide + 5 mM EDTA). Tumor-infiltrating CD4+ Tconv (TCRb+ CD4+ RFP-) cells were sorted with a FACSAria III (BD Biosciences).