分离小鼠原代滋养细胞及滋养细胞侵袭实验
Summary
在这个协议中，我们描述了剥离的胎盘从怀孕d10.5，将鼠标放在隔离的滋养层细胞，用Percoll梯度。然后，我们演示如何使用分离的细胞在基底膜侵袭实验。
Abstract
胎盘是负责运输的养分，气体和生长因子的胎儿，以及消除废物。因此，胎盘发育缺陷的胎儿和母亲有重要的影响，是胚胎致死的一个重要原因。胎盘的胎儿部分是主要的细胞类型的滋养。小学小鼠胎盘滋养层细胞是一种有用的工具，用于研究正常和不正常的胎盘发育，不同的细胞系，可以分离和研究滋养层细胞在特定阶段的怀孕。此外，从转基因小鼠的原代培养的滋养层细胞可用于研究胎盘细胞中的特定基因的作用。这里介绍的是基于该协议索尔达松等人的描述，其中一个Percoll梯度是用来获取一个相对纯净的小鼠离体胎盘滋养层细胞的人口从。这是一个类似的更广泛地使用ð人滋养层细胞的分离方法2-3。分离的细胞的免疫细胞化学染色细胞角蛋白7 4纯度进行评估。在这里，分离的细胞进行分析，然后使用基底膜侵袭实验，以评估滋养细胞侵袭的体外 5-6。入侵细胞和免疫组化染色，计数分析。
Protocol
Discussion
在这段视频中，我们展示了从小鼠胎盘滋养层细胞的分离。然后，我们在体外试验对滋养细胞浸润。此过程中，主要的，但不完全，纯净的滋养层细胞可以得到。磁珠分离或流式细胞术如果需要进一步纯度，可以执行，正如已经描述了初级人类滋养层细胞。适当的长度，的胶原酶分离步骤必须确定每个实验，将学到的经验。消化严重降低细胞的恢复，而下消化可能导致降低纯度，以及减少数字。?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
作者要感谢大学的密苏里学术支持中心制作这个视频的创意服务团队。这项工作是支持的尤尼斯肯尼施莱佛国立儿童健康与人类发展的美国国立卫生研究院，授予数量HD055231
Materials
Specific reagents and equipment:
Wash Solution (filter sterilize after mixing)
| Reagent | Amount | Final concentration |
| 10x Medium 199 | 50ml | 1X |
| Hepes | 2.383 g | 0.02M |
| sodium bicarbonate | 0.42 g | 0.01M |
| Penicllin-Streptomycin | 5 mL | 100 U- 100 μg /ml |
| Sterile dH20 | to 500 mL |
Dissociation solution (make fresh and filter sterilize)
| Reagent | Amount | Final concentration |
| Wash Solution | 100 mL | |
| DNase | 1 vial (2000 U) | 20 U/mL |
| Collagenase | 100 mg | 125 digestion units/mL |
| Name of the reagent | Company | Catalogue number | Comments |
| Collagenase | Sigma | C9891 | This is Clostridium collagenase. Bovine pancreatic collagenase would likely work as well |
| NCTC-135 | Sigma | D4263 | Add FBS to 10% |
| Matrigel invasion chambers | BD Biosciences | 354481 | |
| 10x Medium 199 | Sigma | M9163 | |
| Penicillin-Streptomycin | Invitrogen | 15140 | |
| DNase | Sigma | D4263 | |
| 100 μm Cell strainer | Fisher | 22363549 | |
| Antibody to cytokeratin 7 | DAKO | M7018 | Used at 1:100 dilution |
Equipment
Standard cell culture equipment including a CO2 incubator is required. In addition, a centrifuge rotor capable of spinning 50 ml tubes at 30,000 x g is needed. Note that this exceeds the maximum speed of most conical centrifuge tubes and will likely require an Oak Ridge style tube.
References
- Thordarson, G., Folger, P., Talamantes, F. Development of a placental cell culture system for studying the control of mouse placental lactogen II secretion. Placenta. 8, 573-585 (1987).
- Petroff, M. G., Phillips, T. A., Ka, H., Pace, J. L., Hunt, J. S. Isolation and culture of term human trophoblast cells. Methods. Mol. Med. 121, 203-217 (2006).
- Kliman, H. J., Nestler, J. E., Sermasi, E., Sanger, J. M., Strauss, J. F. Purification, characterization, and in vitro differentiation of cytotrophoblasts from human term placentae. Endocrinology. 118, 1567-1582 (1986).
- Blaschitz, A., Weiss, U., Dohr, G., Desoye, G. Antibody reaction patterns in first trimester placenta: implications for trophoblast isolation and purity screening. Placenta. 21, 733-741 (2000).
- Librach, C. L. 92-kD type IV collagenase mediates invasion of human cytotrophoblasts. J. Cell. Biol. 113, 437-449 (1991).
- Schulz, L. C., Widmaier, E. P. The effect of leptin on mouse trophoblast cell invasion. Biol. Reprod. 71, 1963-1967 (2004).
- Tanaka, S., Kunath, T., Hadjantonakis, A. K., Nagy, A., Rossant, J. Promotion of trophoblast stem cell proliferation by FGF4. Science. 282, 2072-2075 (1998).
