小鼠气管基底细胞和粘膜下腺体的导管细胞分离
Summary
在这里，我们展示了我们的协议，用于隔离的基础和粘膜下腺体导管细胞从小鼠气管。我们还表明，该方法的干细胞注射到背鼠标脂肪垫，以创建一个<em在体内</em>模式，粘膜下腺体的再生。
Abstract
大航空公司直接与环境接触，因此容易受到伤害的毒素和传染性我们呼吸的1。大航空公司，因此需要一个有效的修复机制来保护我们的身体。这种修复过程发生在呼吸道的干细胞，这些干细胞气道隔离理解的修复和再生的机制是非常重要的。同样重要的是理解异常修复，可导致气道疾病的2。该方法的目标是从小鼠气管粘膜下腺导管分离出一种新的干细胞群体，将这些细胞在体外和体内模型系统，以确定的修复和再生机制的粘膜下腺体3。生产表明，可用于分离和检测从大的气道3中的管道和基底的干细胞的方法，这将允许我们研究疾病，如囊性纤维化，哮喘和慢性阻塞性肺疾病的气道。目前，有没有隔离的粘膜下腺体导管细胞的方法，也没有在体内模型研究粘膜下腺体的再生。
Protocol
Discussion
这种技术来隔离气道管和基底细胞重要的是，提高我们对气道的修复和再生，气道疾病。这里描述的技术包括几个关键的步骤。第一是优化酶消化期间。第二个是创造的单细胞悬液通过串行逐步提高计针，以防止传代细胞剪切，但分手的细胞团块。第三个是FACS分析，并适当的抗体的细胞的门控。
由于一些酶带钢表面抗原表位，像ITGA6和NGFR，一个可能的修改是改变内切酶酶切?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们要感谢宽广的干细胞研究中心FACS，尤其要感谢他们的帮助杰西卡·斯科尔斯和Felicia科德雷亚与细胞分选。这项工作是由CIRM RN2-00904-1，K08 HL074229，美国胸科学会/慢性阻塞性肺病基金会ATS-06-065，值得关注的基金会，加州大学洛杉矶分校琼森综合癌症中心胸腔肿瘤计画/肺癌SPORE大学，加州癌症研究协调委员会和格温哈森樱桃纪念实验室（BG）。
Materials
| Name of the reagent | Company | Catalog number |
| Complete medium 10: DMEM-F-12 , 50/50, 1X) |
Mediatech | 15-090-CV |
| Hepes (15 mM) | Invitrogen | 15630 |
| Sodium bicarbonate (3.6mM or 0.03%) | Invitrogen | 25080 |
| L-glutamine (4 mM) | Mediatech | 25-005-Cl |
| Penicillin (100 U/ml) | Mediatech | 30-001-CI |
| Streptomycin (100 μg/m) | Mediatech | 30-001-CI |
| Amphotericin B (0.25 μg/ ml) | Lonza | 17-836R |
| Insulin (10 μg/ml) | Sigma | I6634 |
| Transferrin (5 μg/ml) | Sigma | T1147 |
| Cholera toxin (0.1 μg/ml) | Sigma | C8052 |
| Epidermal Growth Factor (25 ng/ml) | BD | 354001 |
| Bovine Pituitary Extract (30 μg/ml) | Invitrogen | 13028-014 |
| Fetal Bovine Serum (5%) | Fisher | SH3008803HI |
| Retinoic acid (0.05 μM) | Sigma | R2625 |
| Growth Factor Reduced Matrigel | BD | 354230 |
Table 1. Complete media components.
| Name of the reagent | Company | Catalogue number | Comments |
| Pronase | Roche | 10165921001 | Used at 0.15%: -o/n at 4 °C digestion to isolate total tracheal cells (for ALI culture) -4 hr digestion 4 °C to isolate SMG |
| Dispase | BD Biosciences | 354235 | Used at 16 Units: 30 min at RT |
| DNase I | Sigma | DN25 | Used at 0.5 mg/ml: 20-30 min at RT |
Table 2. Enzymes used for enzymatic digestion of the trachea.
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