小鼠呼吸道上皮细胞和实验香烟烟雾暴露在空气分离液界面
Summary
从小鼠的呼吸道和肺上皮细胞可以分离，气液界面的差异化的呼吸道上皮模式培养。协议是分离，培养，这些细胞暴露卷烟主流烟雾，为了研究这种环境毒素的分子反应。
Abstract
肺上皮细胞可以分离出小鼠的呼吸道及气 – 液界面（ALI）的培养作为一个差异化的呼吸道上皮模型。协议是描述卷烟主流烟雾（CS）隔离和揭露这些细胞，上皮细胞的反应，以研究为CS暴露。该协议由三部分组成：隔离的呼吸道上皮细胞从小鼠气管，气 – 液界面（ALI）作为完全分化的上皮细胞，这些细胞的培养，这些细胞在文化和校准主流CS交付。 ALI的文化系统允许的条件下，更接近于他们比普通的液体培养系统的生理设置的呼吸道上皮细胞的文化。接触到CS分子和肺癌细胞反应的研究是了解环境空气污染对人体健康的影响的关键组成部分。最终，在这方面的研究结果可能有助于了解慢性阻塞性肺疾病（COPD），及其他与烟草有关的疾病，这代表了重大的全球性健康问题的病因。
Protocol
Discussion
协议描述小鼠气管上皮细胞的分离，是改编自你等的协议 ，1，和其他修改2-3。至于任何协议，描述细胞的隔离，最关键的环节，以避免细菌或真菌病原体的污染，采用严格的无菌操作技术。第二个关键步骤，以避免成纤维细胞污染的文化，可避免的气管仔细解剖，和阴性选择步骤1.19。文化监控，洗净，和文化传媒补充隔日最初的72小时孵化后，应充分区分文化在大约两个?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们感谢技术援助和博士Shivraj Tyagi埃梅卡Ifedigbo宝贵的专业知识。我们也感谢哈佛NeuroDiscovery中心与显微镜的帮助。这项工作是支持部分由美国心脏协会授予09PRE2250120 Predoctoral林Hilaire指出，与美国国立卫生研究院拨款，以R01 – HL60234，R01 – HL55330，R01 – HL079904，授予AMK的财。
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Ham’s F12 Medium 1X | Cellgro | MT-10-080-CM | With L-glutamine | |
| Pen/strep | Lonza | 17-602E | ||
| Pronase | Roche | 10165921001 | Streptomyces griseus | |
| Collagen I | BD biosciences | 354236 | From rat tail | |
| Acetic Acid | Sigma | 338826-25 | ||
| DNaseI | Sigma | DN25-100MG | From Bovine Pancreas | |
| Bovine Serum Albumin | Fisher Scientific | BP1605-100 | Fraction V | |
| Retinoic Acid | Retinoic Acid | R265-50MG | ||
| Hank’s Balanced Salt Solution | Gibco | 14175 | Without Ca++ or Mg++ | |
| DMEM-F12 | Cellgro | MT-15-090-CM | Without L-Glutamine or HEPES | |
| HEPES, 1M in H2O | Sigma | 83264-100ML | ||
| L-Glutamine | Sigma | G7513-100ML | 200 mM | |
| Amphotericin B (Fungizone) | Fisher Scientific | 1672346 | ||
| Insulin | Sigma | 16634-50MG | Bovine Pancreas | |
| Apo-transferrin (human) | Sigma | T1147-100MG | ||
| Cholera toxin | Sigma | C8052 | Vibrio Cholerae | |
| Epidermal growth factor | BD Biosciences | 354001 | Mouse | |
| Bovine pituitary Extract | BD Biosciences | 354123 | ||
| NuSerum | BD Biosciences | 355100 | ||
| Transwell | Corning Costar | 3401 | 12 mm, 0.4 mm Pore Polycarbonate |
|
| Primaria 100 mm culture dish | Falcon | 353803 | ||
| Pallflex membrane | Pall Life Sciences | EMFAB TX40H120-WW | ||
| Smoking Machine | EMI Services | ATCSALI-1 | see Footnote* |
*The cigarette smoking machine is a custom designed and fabricated 14″x14″x20″ Dual chambered and water jacketed light tint clear proof 1/2″ thick polycarbonate Lexan chamber for cigarette smoke exposure with temperature controlled, water level sensor controlled shut off system. A cigarette smoking/puffing unit is installed for a variable cigarette puffing rates. When the unit is in use, it mimics an incubator in the sense that the temperature, humidity and carbon dioxide are controlled in the system. The system includes: (I) a customized dual chamber/water jacketed unit that maintains a controlled environment for tissue culture experiments. (II) A digital heavy duty, high precision dual pump water temperature circulator system with water level sensor and temperature control (III) A cigarette smoking unit with puffing pump. (IV) A pump cycle sensor control rate cycler (IV) A Stainless steel high precision in-Line filter holder. (V) A Detachable lid mounted 11/2″ size axial uniformity cigarette smoke mixing fan. (VI) A medium size water bath with mounting bracket for the water circulator. (VII) A 1/2′ thick Plexiglas tray with brackets for the puff pump and holder for cigarette ash collector.
This machine as described can be substituted with similar commercially-available smoking machines such as the kind available from TSE systems (www.tse-systems.com).
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