Isolation of Murine Alveolar Type II Epithelial Cells
小鼠肺泡II型上皮细胞分离
发布: 2017年05月20日第7卷第10期 DOI: 10.21769/BioProtoc.2288 浏览次数: 11672
评审: HongLok LungJason A. NeidlemanShahzada Khan
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参见作者原研究论文
The authors used this protocol in:
May 2016
Abstract
We have optimized a protocol for isolation of alveolar type II epithelial cells from mouse lung. Lung cell suspensions are prepared by intratracheal instillation of dispase and agarose followed by mechanical disaggregation of the lungs. Alveolar type II epithelial cells are purified from these lung cell suspensions through magnetic-based negative selection using a Biotin-antibody, Streptavidin-MicroBeads system. The purified alveolar type II epithelial cells can be cultured and maintained on fibronectin-coated plates in DMEM with 10% FBS. This protocol enables specific investigation of alveolar type II epithelial cells at molecular and cellular levels and provides an important tool to investigate in vitro the mechanisms underlying lung pathogenesis.
Keywords: Alveolar type II epithelial cells (肺泡Ⅱ型上皮细胞)
Lung (肺)
Biotin (生物素)
Streptavidin (链亲和素)
Dispase (分散酶)
Agarose (琼脂糖)
Background
Alveolar type II epithelial cells play critical roles in alveolar integrity maintenance, surfactant protein synthesis and secretion, and defense against pulmonary infection of bacteria and viruses. Recent studies using mouse lung cancer models have proven that alveolar type II epithelial cells are a key cell of origin of adenoma/adenocarcinoma induced by chemical carcinogens and oncogenic mutations (Qu et al., 2015; Zhou et al., 2015 and 2017). To further expand our understanding of the role of alveolar type II epithelial cells in lung pathogenesis in vivo, isolation of alveolar type II epithelial cells is needed to allow for a precise mechanism analysis in vitro. Based on previous studies (Corti et al., 1996; Rice et al., 2002), a modified method was used in our laboratory to isolate highly purified, viable and culturable alveolar type II epithelial cells from mice (Zhou et al., 2015; Sun et al., 2016).
Materials and Reagents
- Needles (BD, catalog number: 305167 ) or tapes
- 10 ml syringe (BD, catalog number: 309604 )
- 27 gauge needle (BD, catalog number: 305109 )
- Nylon string (Dynarex, catalog number: 3243 )
- 22 G x 1” Exel Safelet Catheter (Exel International, catalog number: 26746 )
- 1 ml syringe (BD, catalog number: 309659 )
- 15 ml tubes (VWR, catalog number: 89039-666 )
- 60 mm non-coated cell culture dish (Greiner Bio One International, catalog number: 628160 )
- Cell strainer (70 µm) (Fisher Scientific, catalog number: 22-363-548 )
- Cell strainer (40 µm) (Fisher Scientific, catalog number: 22-363-547 )
- Nylon mesh (25 µm) (ELKO filtering, catalog number: 03-25/19 )
- MS column (Miltenyi Biotec, catalog number: 130-042-201 )
- Fibronectin-coated plate (Corning, catalog number: 354402 )
- Mice (THE JACKSON LABORATORY)
- 70% ethanol (Decon Labs, catalog number: 2701 )
- Dispase (1 mg/ml dissolved in PBS) (Roche Diagnostics, catalog number: 4942078001 )
- 1% low melting point agarose (Dissolved in PBS, autoclaved, aliquoted and stored at 4 °C) (Lonza, catalog number: 50100 )
- DMEM (Lonza, catalog number: 12-604F )
- DNase I (Roche Diagnostics, catalog number: 10104159001 )
- Biotinylated anti-CD45 (Miltenyi Biotec, catalog number: 130-101-952 )
- Biotinylated anti-CD16/CD32 (Miltenyi Biotec, catalog number: 130-101-895 )
- Streptavidin MicroBeads (Miltenyi Biotec, catalog number: 130-048-101 )
- Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10437028 )
- Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9625 )
- Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
- Disodium hydrogen phosphate heptahydrate (Na2HPO4·7H2O) (Fisher Scientific, catalog number: BP331-500 )
- Potassium phosphate monobasic (KH2PO4) (Acros Organics, catalog number: 205925000 )
- Bovine serum albumin (BSA) (MP Biomedicals, catalog number: 199898 )
- Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E26282-500G )
Note: This product has been discontinued. - Penicillin-streptomycin (Lonza, catalog number: 17-602E )
- Phosphate buffered saline (PBS) (see Recipes)
- Labeling buffer (see Recipes)
Equipment
- CO2 chamber
- Biosafety cabinet
- Styrofoam board
- Forceps (Roboz Surgical Instrument, catalog number: RS-5135 )
- Scissors (Roboz Surgical Instrument, catalog number: RS-6802 )
- Water bath incubator (Thermo Fisher Scientific, Thermo ScientificTM, model: BarnsteadTM 18000A-1CE )
- Shaker (Thermo Fisher Scientific, Thermo ScientificTM, model: BarnsteadTM 2314 )
- Centrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: IEC CL40R , catalog number: 11210927)
- MACS MultiStand (Miltenyi Biotec, catalog number: 130-042-303 )
- Hemocytometer (Hausser Scientific, catalog number: 3110 )
Procedure
文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Sun, F., Xiao, G. and Qu, Z. (2017). Isolation of Murine Alveolar Type II Epithelial Cells. Bio-protocol 7(10): e2288. DOI: 10.21769/BioProtoc.2288.
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分类
癌症生物学 > 通用技术 > 细胞生物学试验
细胞生物学 > 细胞分离和培养 > 细胞生长
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