活体成像小鼠皮下瘤模型实验步骤
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活体成像技术能够让研究人员直接快速的测量各种癌症模型中肿瘤的生长、转移以及对药物的反应。其特点是极高的灵敏度使微小的肿瘤病灶（少到几百个细胞）也可以被检测到，比传统方法的灵敏度大大提高了；非常适合于肿瘤体内生长的定量分析；避免由于宰杀老鼠而造成的组间差异；节省动物成本。本文阐述活体成像实验中小鼠皮下瘤模型实验操作步骤。
工具/原料
- MDA-MB-231-Luc and HCT116-Luc cells
- Mice: 2 subcutaneous HCT116-Luc tumor-bearing female BALB/c nude mice; 4 naïve female BALB/c nude mice;
- Artagain black paper (Strathmore, Catalog# 445-109)
- D-Luciferin Firefly, potassium salt（Sciencelight, Catalog# luc001）, for in vivo imaging
- Syringe (1ml) and needles (25 x 5/8" gauge)
- Anesthesia (Pentobarbital Sodium)
方法/步骤
Luciferin Preparation
1. Prepare a stock solution of luciferin at 15mg/ml in DPBS. Filter sterilize through a 0.2 um filter.
2. Prepare enough to inject 10 ul/g of body weight. Each mouse should receive 150 mg luciferin/kg body weight. (e.g. For a 20 g mouse, inject 200 ul to deliver 3 mg of luciferin.)
Preparation of tumor cells:
1. Trypsinize the cells from flasks, wash once in serum-free medium.
2. Resuspend in room temperate serum-free medium to a concentration of 1x106 cells in 100ul. Hold at room temperature until ready for use.
Injections:
1. Inject mice with firefly luciferin (150 mg/kg) by intraperitoneal injection using a 25 x 5/8” gauge needle.
2. After 7-8 minutes, anesthetize mice by intraperitoneal injection of pentobarbital sodium using a 25 x 5/8” gauge needle.
3. (1) Inject tumor cells (1x106 cells in 100ul) subcutaneously into the dorsal flank of the mouse.
(2) For the two HCT116-Luc tumor-bearing mice, no cells were inoculation to the mice.
Imaging
Place mice on black paper in the imaging box and image dorsally to detect tumor cells.
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注意事项
- 要先注射D-Luciferin Firefly, potassium salt，后麻醉小鼠
- 在注射D-Luciferin Firefly, potassium salt 后15-35分钟之间检测
- 小鼠周龄要在4-6周
- 接种部位要合适