Quantitative colorimetric calcium assay kits

Age-matched, non-obese, diabetic-severe, combined immunodeficient gamma (NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ JAX^®, NOD-SCID γ) male mice (8 weeks old, 20–25 g body weight) were used. 5 × 10⁶ of SNU-308 cells were resuspended in 100 μL of PBS and injected into the subcutaneous. All animal experiments were conducted in accordance with a protocol approved by the Academia Sinica Institutional Animal Care and Utilization Committee (IACUC 14-03-665). One week after tumor injection, different treatment were started. Four groups were included in this study, i.e., ethanol treatment group as the shame group (twice per week, intraperitoneal injection, n = 6), 1α,25(OH)₂D₃ treatment group (0.3 ug/kg, twice per week, intraperitoneal injection, n = 6), MART-10 low-dose group (0.15 ug/kg, twice per week, intraperitoneal injection, n = 6), and MART-10 high-dose group (0.3 ug/kg, twice per week, intraperitoneal injection, n = 6). The tumor volumes, body weight and blood calcium were measured weekly. Tumor masses were harvested after 5 weeks. Blood calcium was measured using quantitative colorimetric calcium assay kits (BioChain, Newark, CA, USA) according to the manufacturer’s protocol.

The study was approved by the Chang Gung University Animal Research Committee (Permit Number: 2014022601). Young male BALB/C Nu/Nu mice, age 6 weeks old and average weight 20–30 g, were purchased from National Taiwan Animal Center. A total of 24 adult male Young male BALB/C Nu/Nu mice were used in this experiment. 5 × 10⁶ of SNU-308 cells were resuspended in 100 μL of PBS and injected into the subcutaneous. One week after tumor injection, different treatments were started. Four groups were included in this study, i.e., ethanol treatment group as the shame group (twice per week, intraperitoneal injection, n = 6), 1α,25(OH)₂D₃ treatment group (0.3 μg/kg, twice per week, intraperitoneal injection, n = 6), 25(OH)D low-dose group (6 μg/kg, twice per week, intraperitoneal injection, n = 6), and 25(OH)D high-dose group (20 μg/kg, twice per week, intraperitoneal injection, n = 6). The body weight and blood calcium were measured weekly. Tumor masses were harvested after 5 weeks. Blood calcium was measured using quantitative colorimetric calcium assay kits (BioChain, Newark, CA, USA) according to the manufacturer’s protocol.