C57BL/6 strain were used as experimental model to study effect of Ts-depletion in syngeneic LL/2 cells to prevent immune rejection.13 (link) Mice were anaesthetised using isoflurane and euthanised by cervical dislocation.
For subcutaneous injections, 1 × 106 cells resuspended in 50 µl 0.9% NaCl were mixed with Matrigel (Corning) in a ratio 1:1 (v:v). Cells were injected in right flanks of 10–15-weeks-old female C57BL/6, with eight mice per group. Calliper measurements were taken every 4th day and tumour volume was calculated using the formula (Length × Width2 × π)/6.
For tail-vein metastasis assay, 5 × 105 LL/2 pLKO and shTs cells were resuspended in 100 µl PBS and injected in the tail vein of female C57BL/6, with 10 mice per group. Lung metastases were monitored by bioluminescence imaging (BLI) 4 weeks after injection. Anesthetised mice were intraperitoneally injected with 150 mg/kg D-luciferin (Kayman Chemicals). Bioluminescence images were acquired with Lumina III in vivo Imaging System (IVIS, Perkin Elmer). For all the mice exposure time was maintained at 180 s. Raw IVIS images were analysed with Living Image software and the metastasis was represented as radiance.
For subcutaneous injections, 1 × 106 cells resuspended in 50 µl 0.9% NaCl were mixed with Matrigel (Corning) in a ratio 1:1 (v:v). Cells were injected in right flanks of 10–15-weeks-old female C57BL/6, with eight mice per group. Calliper measurements were taken every 4th day and tumour volume was calculated using the formula (Length × Width2 × π)/6.
For tail-vein metastasis assay, 5 × 105 LL/2 pLKO and shTs cells were resuspended in 100 µl PBS and injected in the tail vein of female C57BL/6, with 10 mice per group. Lung metastases were monitored by bioluminescence imaging (BLI) 4 weeks after injection. Anesthetised mice were intraperitoneally injected with 150 mg/kg D-luciferin (Kayman Chemicals). Bioluminescence images were acquired with Lumina III in vivo Imaging System (IVIS, Perkin Elmer). For all the mice exposure time was maintained at 180 s. Raw IVIS images were analysed with Living Image software and the metastasis was represented as radiance.
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