All animal procedures were in accordance with the National Institutes of Health guidelines and approved by the Institutional Animal Care and Use Committee of Washington State University. 8- to 12-week old male wild-type and Per1/2−/− mice on C57BL/6 background [36 (link)] were obtained from the Jackson Laboratories. The mice were maintained under a 12 h/12 h light/dark cycle (light on at 7 AM, ZT0, and off at 7 PM, ZT12) at least 4 weeks before and through the duration of the study.
For tumor studies, B16F10 melanoma cells were purchased from ATCC and cultured in RPMI-1640 + 10% FBS. 2×105 cells in 50% matrigel (Corning) were injected into the lower right flank region of each mouse. Body weights were measured using an analytical balance, and tumor volumes were measured using a digital caliper and calculated using the formula: V = (W2 x L)/2 [63 (link)]. Tumor-bearing mice were sacrificed as tumor volumes crossed 4X the volume at the start of cisplatin treatment (for toxicity and tumor study) or reached an average of 650 mm3 on day 15 (for immunophenotyping). Upon sacrifice, blood, kidney, spleen, lymph nodes, testis, brain, and tumor tissues were harvested for further analysis.
For tumor studies, B16F10 melanoma cells were purchased from ATCC and cultured in RPMI-1640 + 10% FBS. 2×105 cells in 50% matrigel (Corning) were injected into the lower right flank region of each mouse. Body weights were measured using an analytical balance, and tumor volumes were measured using a digital caliper and calculated using the formula: V = (W2 x L)/2 [63 (link)]. Tumor-bearing mice were sacrificed as tumor volumes crossed 4X the volume at the start of cisplatin treatment (for toxicity and tumor study) or reached an average of 650 mm3 on day 15 (for immunophenotyping). Upon sacrifice, blood, kidney, spleen, lymph nodes, testis, brain, and tumor tissues were harvested for further analysis.
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