Isolation of Highly Pure Primary Mouse Alveolar Epithelial Type II Cells by Flow Cytometric Cell Sorting
采用流式细胞术分选术分离高纯度原代小鼠II型肺泡上皮细胞
发布: 2016年11月20日第6卷第22期 DOI: 10.21769/BioProtoc.2013 浏览次数: 27665
评审: Fanglian HeHongwei HanAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Jun 2016
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Abstract
In this protocol, we describe the method for isolating highly pure primary alveolar epithelial type II (ATII) cells from lungs of naïve mice. The method combines negative selection for a variety of lineage markers along with positive selection for EpCAM, a pan-epithelial cell marker. This method yields 2-3 x 106 ATII cells per mouse lung. The cell preps are highly pure and viable and can be used for genomic or proteomic analyses or cultured ex vivo to understand their roles in various biological processes.
Background
The internal surfaces of lungs are lined by epithelial cells, the type of epithelial cell varying morphologically and functionally with the location within the lung. ATII cells are one of the two types of epithelial cells that line the alveolar walls and have been described to play critical roles in surfactant synthesis and secretion. They are also part of the first line of defense within the lung and are involved in initiating and modulating immune responses during pulmonary infection or allergy. They are also thought to act as progenitor cells in the distal lung with proliferative capacity and the ability to repair the epithelium after injury. Available methods of ATII isolation did not yield cell preps that were more than 80-85% pure, making them unsuitable for reliable analyses of mRNA and protein expression. The method described here is an improvement over prior methods and yields mouse primary ATII cell preps with the highest purity that can thus be reliably used for expression analyses. For further discussion on the method, we refer the reader to the original publication from where this protocol originates (Sinha et al., 2016).
Materials and Reagents
- C57BL/6 mice (Charles River Laboratories, strain code: 027)
- 2,2,2-tribromoethanol (Avertin) (Sigma-Aldrich, catalog number: T48402 )
- tert-Amyl alcohol (Sigma-Aldrich, catalog number: 240486 )
- 70% ethanol (Decon Labs, catalog number: V1401 )
- 1 ml slip-tip syringe (important to not use a Luer-LokTM tip syringe) (BD, catalog number: 309659 )
- 10 ml syringe (BD, Luer-LokTM, catalog number: 309604 )
- 25 G x 5/8 inch needle (BD, catalog number: 305122 )
- 20 G x 1.16 inch angiocatheter (BD, AngiocathTM, catalog number: 381134 )
- Surgical silk suture 3-0 (Henry Schein, catalog number: 100-7842 )
- 70 μm cell strainer (Corning, catalog number: 431751 )
- 40 μm cell strainer (Corning, catalog number: 352340 )
- 20 μm nylon mesh (Spectrum, catalog number: 146510 )
- Kimwipes (Thermo Fisher Scientific, Fisher Scientific, catalog number: 06-666A )
- 5 ml polypropylene tubes (Corning, Falcon®, catalog number: 352063 )
- 15 ml tubes (Corning, catalog number: 430052 )
- 50 ml conical tube (Corning, catalog number: 430290 )
- Non TC-treated 10 cm Petri dish (Corning, catalog number: 430591 )
- D-PBS (without Ca2+ and Mg2+, no phenol) (Thermo Fisher Scientific, catalog number: 14190250 )
- 0.5 M EDTA, pH 8.0 (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15575020 )
- Dispase II (neutral protease, grade II) (Sigma-Aldrich, catalog number: 4942078001 )
- Low melting point agarose (Sigma-Aldrich, catalog number: A9419 )
- DMEM (Thermo Fisher Scientific, GibcoTM, catalog number: 11965-092 )
- Penicillin-streptomycin (10,000 U/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
- 1 M HEPES (Thermo Fisher Scientific, GibcoTM, catalog number: 15630080 )
- ACK lysis buffer (Lonza, catalog number: 10-548E )
- Biotinylated anti-mouse CD45, clone 30-F11, 0.5 mg/ml (Affymetrix, eBioscience, catalog number: 13-0451-85 )
- Biotinylated anti-mouse CD16/32, clone 2.4G2, 0.5 mg/ml (BD, PharmingenTM, catalog number: 553143 )
- Biotinylated anti-mouse CD31, clone MEC13.3, 0.5 mg/ml (Biolegend, catalog number: 102504 )
- Biotinylated anti-mouse TER119, clone TER119, 0.5 mg/ml (Affymetrix, eBioscience, catalog number: 13-5921-85 )
- Biotinylated anti-mouse Integrin β4, clone 346-11A, 0.5 mg/ml (Biolegend, catalog number: 12603 )
- Anti-mouse EpCAM-APC, clone G8.8, 0.2 mg/ml (Affymetrix, eBioscience, catalog number: 17-5791-82 )
- Dynabeads® MyOneTM streptavidin T1 magnetic beads (Thermo Fisher Scientific, InvitrogenTM, catalog number: 65601 )
- Streptavidin-PE, 0.5 mg/ml (BD, PharmingenTM, catalog number: 554061 )
- Rabbit anti-pro-SP-C, polyclonal serum (EMD Millipore, catalog number: AB3786 )
- DNase I (Sigma-Aldrich, catalog number: D5025 )
- Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 26140 )
- DAPI (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: D1306 )
- Avertin solution (100% stock working solution) (see Recipes)
- Avertin solution (2.5% stock working solution) (see Recipes)
- Dispase solution (see Recipes)
- 1% Low melting agarose (see Recipes)
- Complete DMEM (see Recipes)
- DNase I solution (see Recipes)
- Sort buffer (see Recipes)
Equipment
- Fine-tipped small scissors (Roboz surgical instrument company, catalog number: RS-5910 )
- Fine forceps (Roboz surgical instrument company, catalog number: RS-5211 )
- Horizontal platform orbital rocker (VWR, catalog number: 40000-300 )
- Tube rotator (Thermo Fisher Scientific, catalog number: 400110Q )
- Orbital incubator-shaker (VWR, catalog number: 10027-214 )
- Biosafety cabinet (VWR, catalog number: 89413-126 )
- Stratalinker (Stratagene, model: 1800 )
- DynaMagTM-2 magnetic separator (Thermo Fisher Scientific, catalog number: 12321D )
- FACS Aria cell sorter (BD, model: BD FACSARIA III )
Software
- FACS Diva (BD Biosciences)
- FlowJo (TreeStar)
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Sinha, M. and Lowell, C. A. (2016). Isolation of Highly Pure Primary Mouse Alveolar Epithelial Type II Cells by Flow Cytometric Cell Sorting. Bio-protocol 6(22): e2013. DOI: 10.21769/BioProtoc.2013.
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分类
免疫学 > 免疫细胞分离 > 维持和分化
免疫学 > 免疫细胞染色 > 流式细胞术
细胞生物学 > 细胞分离和培养 > 细胞分离
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