D luciferin

Mice were injected subcutaneously with 4T1-Luc2 cells (5 × 10⁵/100 μl PBS/mouse) into the fourth mammary fat pad under isoflurane anesthesia. Tumor growth was monitored by measuring the tumor volume according to the formula: volume = length × (width)²/2. After tumors were established (180-200 mm³) on day 15, test mice were divided into different groups (8 mice/group) and subjected to different treatments. At 15 days post tumor cell implantation, primary 4T1 tumors in situ were surgically removed by a tumor resection process. For immunization with test TCL vaccines, mice were then administered with control (PBS) or specific TCL (100 μg/200 μl/mouse) by intravenous injection for 3 weeks (3 injections/week) post tumor resection. To monitor progression of metastatic tumors, control (PBS injection only) and different TCL-treated mice were compared for their tumor metastatic activity and survival rate in another 8 weeks. For immunization with DC vaccines, mice were administered with specific or control DC vaccines (1 × 10⁶ DCs/200 μl PBS/mouse) by intravenous injection at 1, 8 and 15 days post tumor resection. Bioluminescence signals from the 4T1-luc2 tumor cells in test mice were analyzed using a non-invasive IVIS imaging system (Calipers, Hopkinton, MA) after intraperitoneal injection of 150 mg/kg D-luciferin (NanoLight Technology, Pinetop, AZ).

Mice were injected subcutaneously with 4 T1-Luc2 cells (5 × 10⁵/50 μl PBS/mouse) into the fourth mammary fat pad under isoflurane anesthesia. Tumor growth was monitored by measuring the tumor volume, as length × (width)²/2. After tumors were established (180–200 mm³) on day 15, test mice were divided into different groups (8 mice/group) and the primary tumors were surgically resected. For immunization with DC vaccines, mice were subjected to control treatment (PBS) or with specific DC vaccines (1 × 10⁶/200 μl PBS/mouse) via intravenous injection at 0, 7 and 14 days post tumor resection. To monitor the progression of metastatic tumors, bioluminescence signals from the 4 T1-luc2 tumor cells in test mice were analyzed using a non-invasive IVIS imaging system (Calipers, Hopkinton, MA) after intraperitoneal injection of 150 mg/kg D-luciferin (NanoLight Technology, Pinetop, AZ). For detection of myeloid-derived suppressor cells (MDSCs), peripheral blood cells from test mice were collected at 10 days post the secondary boosting of DC vaccine or PBS (control). The criteria used to determine humane endpoint was strictly based on the Guidelines for Determining Endpoints and Humane Termination of Animals provided by the Institutional Animal Care and Use Committee (IACUC) of Academia Sinica, Taiwan. All test mice were euthanatized by CO2 inhalation at the end of experiments.