All mice were maintained under specific pathogen-free conditions and experimental procedures were performed according to the institutional animal welfare guidelines approved by Institutional Animal Care and Use Committee for the University of Kansas Medical Center.
For subcutaneous tumour growth assays, cells were dissociated into single-cell suspensions using non-enzymatic cell dissociation solution (Sigma Biochemicals) and numbers of live cells were counted following trypan blue staining (Thermo Fisher Scientific). Cell suspension in 50 μl of 4.5 mg ml−1 Matrigel (BD Biosciences) in Hank's balanced salt solution was subcutaneously injected into flanks of NIH-III nude mice (Charles River). Tumours were measured three dimensionally two to three times a week for 17–20 days. For tail vein assays, 150 μl of cell suspension (5 × 104) was injected into the lateral veins of nude mice. Mice were monitored for laboured breathing and the numbers of pulmonary tumour nodules were evaluated 6 weeks after injections. For orthotopic injections, 15 μl of cell suspension (1 × 105) was injected into femoral bone marrow space of anaesthetized NOD-scid IL2Rγnull mice (The Jackson Laboratories)22 (link). When the tumours reached ∼1 cm in thigh diameter, the mice were killed. The weight of the primary tumours and numbers of tumour nodules in the lungs and liver (>0.5 mm) were measured.
For subcutaneous tumour growth assays, cells were dissociated into single-cell suspensions using non-enzymatic cell dissociation solution (Sigma Biochemicals) and numbers of live cells were counted following trypan blue staining (Thermo Fisher Scientific). Cell suspension in 50 μl of 4.5 mg ml−1 Matrigel (BD Biosciences) in Hank's balanced salt solution was subcutaneously injected into flanks of NIH-III nude mice (Charles River). Tumours were measured three dimensionally two to three times a week for 17–20 days. For tail vein assays, 150 μl of cell suspension (5 × 104) was injected into the lateral veins of nude mice. Mice were monitored for laboured breathing and the numbers of pulmonary tumour nodules were evaluated 6 weeks after injections. For orthotopic injections, 15 μl of cell suspension (1 × 105) was injected into femoral bone marrow space of anaesthetized NOD-scid IL2Rγnull mice (The Jackson Laboratories)22 (link). When the tumours reached ∼1 cm in thigh diameter, the mice were killed. The weight of the primary tumours and numbers of tumour nodules in the lungs and liver (>0.5 mm) were measured.
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