Проточной цитометрии Выделение первичных мышиных Type II альвеолярных эпителиальных клеток для функционального и молекулярных исследований
Summary
Мы описываем быстрого выделения первичных мышиных тип II альвеолярных эпителиальных клеток (AECII) методом проточной цитометрии отрицательного отбора. Эти AECII показывают высокую жизнеспособность и чистоты и подходят для широкого спектра функциональных и молекулярных исследований в отношении их роли в респираторных заболеваний, таких как аутоиммунные и инфекционные заболевания.
Abstract
Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.
Protocol
Representative Results
Discussion
Наш протокол для выделения мышиных AECII с помощью проточной цитометрии предлагает быстрый способ доступа к первичной клетки из легких мышей целый ряд функциональных и молекулярных исследований. Описанная процедура дает весьма жизнеспособной и чистых популяций AECII, достаточные количе?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
Мы хотели бы поблагодарить М. Höxter для оказания технической помощи в сортировке первичных мышиных AECII от уровня биобезопасности 2 пробы.
Эта работа была поддержана грантами от Немецкого исследовательского фонда (DFG) в БД (SFB587, TP 12 и BR2221/1-1) и стипендию от Ганновера биомедицинских исследований Школы (DFG GSC 108) до AA. DB поддерживает инициативу Президента и сети Фонда имени Гельмгольца немецких научно-исследовательских центров (HGF) по контракту W2/W3-029 номер.
Materials
| Name of reagent | Company | Catalogue number | Comments |
| indwelling cannula Introcan 22G | Braun | REF 4252098B | |
| Dispase, 100 ml(5000 caseinolytic units) | BD Biosciences | 354235 | aliquot to 4 ml in 15 ml tubes, store at -20 °C |
| Biozym Plaque Agarose | Biozym | 840101 | 1% w/v in H2O |
| Deoxyribonuclease I from bovine pancreas, 2000 Kunitz units/vial | Sigma-Aldrich | D4263 | freshly dissolve content of 1 vial in 300 μl DMEM |
| DMEM | Gibco | 22320-022 | used as provided by manufacturer (Low Glucose, Pyruvate, HEPES) |
| cell strainers (100 μm, 75 μm) | BD Falcon | 352360, 352350 | |
| nylon mesh(48 μm, 30 μm) | Bückmann GmbH | 03-48/26-1020, 03-30/18-108 | |
| CellTrics 50 μm filter | PARTEC | 04-0042-2317 | |
| anti-mouse CD16/CD32 | BioLegend | 101302 | clone 93; purified |
| anti-mouse F4/80 | BioLegend | 123116 | clone BM8; APC coupled |
| anti-mouse CD11b | BioLegend | 101208 | clone M1/70; PE coupled |
| anti-mouse CD11c | BioLegend | 117310 | clone N418; APC coupled |
| anti-mouse CD45 | BioLegend | 103102 | clone 30-F11; purified |
| anti-mouse CD19 | eBioscience | 12-0193-83 | eBio 1D3; PE coupled |
| polyclonal goat anti-rat IgG | BD Pharmingen | 550767 | polyclonal, PE coupled |
Antibodies coupled to alternative fluorochromes can be used, depending on the flow cytometer and lasers available. |
Riferimenti
- Fehrenbach, H. Alveolar epithelial type II cell: defender of the alveolus revisited. Respir. Res. 2, 33-46 (2001).
- Folkerts, G., Nijkamp, F. P. Airway epithelium: more than just a barrier. Trends Pharmacol. Sci. 19, 334-341 (1998).
- Gereke, M., et al. Phenotypic alterations in type II alveolar epithelial cells in CD4+ T cell mediated lung inflammation. Respir. Res. 8, 47 (2007).
- Gereke, M., Jung, S., Buer, J., Bruder, D. Alveolar type II epithelial cells present antigen to CD4(+) T cells and induce Foxp3(+) regulatory T cells. Am. J. Respir. Crit Care Med. 179, 344-355 (2009).
- Gribar, S. C., Richardson, W. M., Sodhi, C. P., Hackam, D. J. No longer an innocent bystander: epithelial toll-like receptor signaling in the development of mucosal inflammation. Mol. Med. 14, 645-659 (2008).
- Herold, S., et al. Alveolar epithelial cells direct monocyte transepithelial migration upon influenza virus infection: impact of chemokines and adhesion molecules. J. Immunol. 177, 1817-1824 (2006).
- Knight, D. A., Holgate, S. T. The airway epithelium: structural and functional properties in health and disease. Respirology. 8, 432-446 (2003).
- Schmiedl, A., Kerber-Momot, T., Munder, A., Pabst, R., Tschernig, T. Bacterial distribution in lung parenchyma early after pulmonary infection with Pseudomonas aeruginosa. Cell Tissue Res. 342, 67-73 (2010).
- Loveday, E. K., Svinti, V., Diederich, S., Pasick, J., Jean, F. Temporal- and Strain-Specific Host MicroRNA Molecular Signatures Associated with Swine-Origin H1N1 and Avian-Origin H7N7 Influenza A Virus Infection. J. Virol. 86, 6109-6122 (2012).
- Marriott, H. M., et al. Interleukin-1beta regulates CXCL8 release and influences disease outcome in response to Streptococcus pneumoniae, defining intercellular cooperation between pulmonary epithelial cells and macrophages. Infect. Immun. 80, 1140-1149 (2012).
- Mata, M., Morcillo, E., Gimeno, C., Cortijo, J. N-acetyl-L-cysteine (NAC) inhibit mucin synthesis and pro-inflammatory mediators in alveolar type II epithelial cells infected with influenza virus A and B and with respiratory syncytial virus (RSV). Biochem. Pharmacol. 82, 548-555 (2011).
- Zarbock, R., et al. The surfactant protein C mutation A116D alters cellular processing, stress tolerance, surfactant lipid composition, and immune cell activation. BMC. Pulm. Med. 12, 15 (2012).
- Taubenberger, J. K., Morens, D. M. The pathology of influenza virus infections. Annu. Rev. Pathol. 3, 499-522 (2008).
- Dobbs, L. G. Isolation and culture of alveolar type II cells. Am. J. Physiol. 258, L134-L147 (1990).
- Corti, M., Brody, A. R., Harrison, J. H. Isolation and primary culture of murine alveolar type II cells. Am. J. Respir. Cell Mol. Biol. 14, 309-315 (1996).
- Beers, M. F., Kim, C. Y., Dodia, C., Fisher, A. B. Localization, synthesis, and processing of surfactant protein SP-C in rat lung analyzed by epitope-specific antipeptide antibodies. J. Biol. Chem. 269, 20318-20328 (1994).
- Phelps, D. S., Floros, J. Localization of pulmonary surfactant proteins using immunohistochemistry and tissue in situ hybridization. Exp. Lung Res. 17, 985-995 (1991).
- Wang, J., et al. Differentiated human alveolar type II cells secrete antiviral IL-29 (IFN-lambda 1) in response to influenza A infection. J. Immunol. 182, 1296-1304 (2009).
- Wang, J., et al. Innate immune response to influenza A virus in differentiated human alveolar type II cells. Am. J. Respir. Cell Mol. Biol. 45, 582-591 (2011).
