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原代細(xì)胞分離中，針對(duì)肺細(xì)胞組織涉及到內(nèi)皮，肺、肺泡Ⅱ型、腺泡，肺腫瘤，肺泡上皮和成纖維細(xì)胞等等的解離通常會(huì)使用酶，如何選擇合適的酶來(lái)完成對(duì)應(yīng)肺細(xì)胞組織的解離，以及原代細(xì)胞采集的常規(guī)操作， 艾美捷針對(duì)肺細(xì)胞相關(guān)的組織分類(lèi)解離分種屬、不同的細(xì)胞、酶組合推薦，推薦濃度、培養(yǎng)基以及參考文獻(xiàn)，詳細(xì)匯總指南：
本技術(shù)指南描述了標(biāo)準(zhǔn)實(shí)驗(yàn)室程序； 為建立細(xì)胞分離方案提供了一種合乎邏輯的實(shí)驗(yàn)方法； 并列出了許多組織特異性參考資料，以幫助開(kāi)發(fā)有效的方法。更多其他組織的解離指南，點(diǎn)擊查看：各組織分離指南。
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| 種屬 | 種屬詳情 | 細(xì)胞名稱(chēng) | 酶組合推薦，含推薦濃度 | 培養(yǎng)基 | 參考文獻(xiàn) |
| 牛 | Bovine | 內(nèi)皮，肺 / Endothelial，Pulmonary | Collagenase: 1000 u/ml； Collagenase Type 2: 1000 u/ml | PBS, CMF | Del Vecchio, P., Siflinger-Birnboim, A., Belloni, P., Holleran, L., Lum, H., and Malik, A.: Culture and Characterization of Pulmonary Microvascular Endothelial Cells, In Vitro Cell Dev Biol 28A, 711, 1992 |
| 豚鼠 | Guinea-pig, male, 250-300 g | 肺泡Ⅱ型 / Alveolar type II | Elastase: 40 u/ml | PBS (see reference) | Sikpi, M., Nair, C., Johns, A., and Das, S.: Metabolic and Ultrastructural Characterizationof Guinea-Pig Alveolar Type II Cells Isolated by Centrifugal Elutriation, Biochim Biophys Acta 877, 20, 1986 |
| 豚鼠 | Guinea-pig, male, 100 g | 單細(xì)胞 / Single cells | Trypsin: 0.25% | CF salt solution | Phillips, H.: Dissociation of Single Cells from Lung or Kidney Tissue with Elastase, In Vitro 8, 101, 1972 |
| 人 | Human | 腺泡 / Aveolar | Elastase: 12.9 u/ml | PBS | Bahmed, K., Boukhenouna, S., Karim, L., Andrews, T., Lin, J., Powers, R., Wilson, M., Lin, C., Messier, E., Reisdorph, N., Powell, R., Tang, H., Mason, R., Criner, G. and Kosmider, B.: The Effect of Cysteine Oxidation on DJ-1 Cytoprotective Function in Human Alveolar Type II Cells., Cell Death Dis 10, 638, 2019 |
| 人 | Human | 肺腫瘤 / Lung tumor | Collagenase Type 1: 45-60 u/ml； Collagenase Type 2: 15-20 u/ml； Collagenase Type 4: 45-60 u/ml； Elastase: 0.002%； Deoxyribonuclease I: 0.002% | DMEM/F12 | Quatromoni, J., Singhal, S., Bhojnagarwala, P., Hancock, W., Albelda, S. and Eruslanov, E.: An Optimized Dissagregation Method for Human Lung Tumors That Perserves the Phenotype and Function of the Immune Cells, J Leukoc Biol 97, 201, 2015 |
| 人 | Human | 肺泡上皮 / Alveolar epithelial | Neutral Protease: 2 u/ml； Trypsin: 0.25%； Elastase: 10 u/ml | Bronchial Epithelial Growth Medium | Barkauskas, C., Cronce, M., Rackley, C., Bowie, E., Keene, D., Stripp, B., Randell, S., Noble, P. and Hogan, B.: Type 2 Alveolar Cells are Stem Cells in Adult Lung, J Clin Invest 123, 3025, 2013 |
| 人 | Human | 肺內(nèi)皮 / Pulmonary endothelial | Collagenase Type 2: 0.2% | RPMI | Mackay, L., Dodd, S., Dougall, I., Tomlinson, W., Lordan, J., Fisher, A. and Corris, P.: Isolation and Characterisation of Human Pulmonary Microvascular Endothelial Cells from Patients with Severe Emphysema., Respir Res 14, 23, 2013 |
| 人 | Human | 肺內(nèi)皮 / Lung endothelial | Collagenase Type 2: 0.2% | PBS | Comhair, S., Xu, W., Mavrakis, L., Aldred, M., Asosingh, K. and Erzurum, S.: Human Primary Lung Endothelial Cells in Culture., Am J Respir Cell Mol Biol 46, 723-30, 2012 |
| 人 | Human | 肺 / Lung | Neutral Protease: 2 u/ml； Collagenase/Dispase: 0.1%； Deoxyribonuclease I: 0.01% | DMEM | Fujino, N., Kubo, H., Suzuki, T., Ota, C., Hegab, A., He, M., Suzuki, S., Suzuki, T., Yamada, M., Kondo, T., Kato, H. and Yamaya, M.: Isolation of Alveolar Epithelial Type II Progenitor Cells from Adult Human Lungs., Lab Invest 91, 363, 2011 |
| 人 | Human | 肺，成纖維細(xì)胞 / Lung，F(xiàn)ibroblasts | Trypsin: 0.05% | DMEM | Zhu, Y., Skold, C., Liu, X., Wang, H., Kohyama., T., Wen, F., Ertl, R., and Rennard, S.: Fibroblasts and Monocyte Macrophages Contract and Degrade Three-Dimensional Collagen Gels in Extended Co-Culture, Respir Res 2 (5), 295, 2001 |
| 人 | Human, 16-24 week therapeutic abortions | 肺泡II型，胎兒 / Alveolar type II，F(xiàn)etal | Trypsin: 50 ug/ml | Ham's F-12, Eagle's MEM | Liley, H., Ertsey, R., Gonzales, L., Odom, M., Hawgood, S., Dobbs, L., and Ballard, P.: Synthesis of Surfactant Components by Cultured Type II Cells From Human Lung, Biochim Biophys Acta 961, 86, 1988 |
| 人 | Human | 上皮 / Epithelial | Trypsin: 0.02% | Medium 199 | Lechner, J., Haugen, A., McClendon, I., and Pettis, E.: Clonal Growth of Normal Adult Human Bronchial Epithelial Cells in a Serum-Free Medium, In Vitro 18 (7), 633, 1982 |
| 人 | Human, 12-16 wk old embryo | 成纖維細(xì)胞 / Fibroblasts | Trypsin: 0.01% | Eagle's MEM | Kan, M., and Yamane, I.: In Vitro Proliferation and Lifespan of Human Diploid Fibroblasts in Serum-Free BSA-Containing Medium, J Cell Physiol 111, 155, 1982 |
| 人 | Human fetuses, 80 day (also swine fetuses, 70 day, adult Amer Dutch, 250 day) | 肺 / Lung | Collagenase: 0.01% | HBSS | Hinz, R., and Syverton, J.: Mammalian Cell Cultures for Study of Influenza Virus. I. Preparation of Monolayer Cultures with Collagenase, Proc Soc Exp Biol Med 101, 19, 1959 |
| 小鼠 | Mouse | 成纖維細(xì)胞 / Fibroblasts | Collagenase Type 2: 1.0% Deoxyribonuclease I: 0.001% | HBSS | Qiu, Y., Xu, Y., Wang, M., Miao, M., Zhou, H., Xu, J., Kong, J., Zheng, D., Li, R., Zhang, R., Guo, Y., Li, X., Cui, J., Qin, C. and Zhou , X.: Flavivirus Induces and Antagonizes Antiviral RNA Interference in Both Mammals and Mosquitoes., Sci. Adv. 6, 7989, 2020 |
| 小鼠 | 2-3 month | 肺內(nèi)皮 / Lung endothelial | Collagenase Type 1: 0.3% | DMEM | Wang, J., Niu, N., Xu, S. and Jin, Z.: A Simple Protocol for Isolating Mouse Lung Endothelial Cells., Sci Rep 9, 1458, 2019 |
| 小鼠 | Mouse, 6 week | Alves CarlosRoberto, Souza RaquelSantos de, Charret KarenDos Santos, C?rtes LuziaMonteiro de Castro, Sá-Silva MatheusPereira de, Barral-Veloso Laura, Oliveira LuizFilipe Gon?alves, da Silva FranklinSouza: Understanding serine proteases implications on Leishmania spp lifecycle., Exp Parasitol 184, 67-81, 2018 | |||
| 小鼠 | Mouse, 6 week | 肺泡上皮 / Alveolar epithelial | Collagenase Type 1: 0.1%； Deoxyribonuclease I: 0.1%； Trypsin: 0.01% | DMEM/F-12 | Lewis, K., Hall, J., Kiyotake, E., Christensen, T., Balasubramaniam, V. and Anseth, K.: Epithelial-Mesenchymal Crosstalk Influences Cellular Behavior in a 3D Alveolus-Fibroblast Model System., Biomaterials 155, 124, 2018 |
| 小鼠 | Mouse | 肺泡上皮 / Alveolar epithelial | Neutral Protease: 0.1%； Deoxyribonuclease I: 0.01% | DMEM | Sun, F., Xiao, G. and Qu, Z.: Isolation of Murine Alveolar Type II Epithelial Cells., Bio-protocol 7, , 2017 |
| 小鼠 | Mouse | 肺 / Lung | Collagenase Type 1: 0.15%； Deoxyribonuclease I: 0.04% | PBS | Yu, Y., O'Koren, E., Holten, D., Kan, M., Kojin, D., Nelson, E., Que, L. and Gunn, M.: A Protocol for the Comprehensive Flow Cytometric Analysis of Immune Cells in Normal and Inflammed Murine Non-Lymphoid Tissues, PLoS ONE 10, 1371, 2016 |
| 小鼠 | Mouse | 肺 / Lung | Collagenase Type 2: 0.3%； Deoxyribonuclease I: 0.3% | PBS | Braza, F., Dirou, S., Forest, V., Sauzeau, V., Hassoun, D., Chesne, J., Cheminant-Muller, M., Sagan, C., Magnan, A. and Lemarchand, P.: Mesenchymal Stem Cells Induce Suppressive Macrophages Through Phagocytosis in a Mouse Model of Asthma., Stem Cells 34, 1836-45, 2016 |
| 小鼠 | Mouse, 8-12 week | 上皮，內(nèi)皮 / Epithelial, Endothelial | Collagenase Type 1: 0.5%； Deoxyribonuclease I: 0.1%； Neutral Protease: 0.1% | PBS | Singer, B., Mock, J., D'Alessio, F., Aggarwal, N., Mandke, P., Johnston, L. and Damarla, M.: Flow-Cytometric Method for Simultaneous Analysis of Mouse Lung Epithelial, Endothelial, and Hematopoietic Lineage Cells., Am J Physiol Lung Cell Mol Physiol 310, L796-801, 2016 |
| 小鼠 | Mouse | 肺泡 / Alveolar | Collagenase Type 1: 300 u/ml； Elastase: 4 u/ml； Neutral Protease: 5 u/ml； Deoxyribonuclease I: 200 u/ml | HBSS | Mohapatra, A., Van Dyken, S., Schneider, C., Nussbaum, J., Liang, H. and Locksley, R.: Group 2 Innate Lymphoid Cells Utilize the IRF4-IL-9 Module to Coordinate Epithelial Cell Maintenance of Lung Homeostasis., Mucosal Immunol , , 2015 |
| 小鼠 | Mouse | 肺 / Pulmonary | Collagenase Type 4: 0.16%； Deoxyribonuclease I: 0.1% | see reference | Kim, H., Lee, H., Chang, Y., Pichavant, M., Shore, S., Fitzgerald, K., Ikawura, Y., Israel, E., Bolger, K., Faul, J., DeKruyff, R. and Umetsu, D.: IL-17 Producing Innate Lymphoid Cells and the NLRP3 Inflammasone Facilitate Obescity-associated Airway Hyperractivity, Nat Med 20, 54, 2014 |
| 小鼠 | Mouse, embryonic | 腺泡祖細(xì)胞 / Avelolar progenitor | Elastase: 3 u/ml； Deoxyribonuclease I: 0.33 u/ml | DMEM/F12 | Desai, T., Brownfield, D. and Krasnow, M.: Alveolar Progenitor and Stem Cells in Lung Development, Renewal and Cancer., Nature 507, 190-4, 2014 |
| 小鼠 | Mouse | 肺 / Lung | Collagenase Type 4: 0.3% | PBS | Guimond, D., Cam, N., Hirve, N., Duan, W., Lambris, J., Croft, M. and Tsoukas, C.: Regulation of Immune Responsiveness In Vivo by Disrupting an Early T-Cell Signaling Event Using a Cell-Permeable Peptide., PLoS ONE 8, e63645, 2013 |
| 小鼠 | Mouse | 肺 / Lung | Collagenase Type 4: 0.1%； Deoxyribonuclease I: 0.01% | HBSS | Misharin, A., Morales-Nebreda, L., Mutlu, G., Budinger, G. and Perlman, H.: Flow Cytometric Analysis of Macrophages and Dendritic Cell Subsets in the Mouse Lung., Am J Respir Cell Mol Biol 49, 503-10, 2013 |
| 小鼠 | Mouse, 8-12 week | 肺祖細(xì)胞 / Lung progenitor | Collagenase/Dispase: 0.2%； Neutral Protease: 5 u/ml； Deoxyribonuclease I: 0.0025% | DMEM | Driscoll, B., Kikuchi, A., Lau, A., Lee, J., Reddy, R., Jesudason, E., Kim, C. and Warburton, D.: Isolation and Characterization of Distal Lung Progenitor Cells., Methods Mol Biol 879, 109, 2012 |
| 小鼠 | Mouse | 肺腫瘤 / Lung tumor | Neutral Protease: 50 u/ml； Collagenase: 400 u/ml； Deoxyribonuclease I: 50 u/ml | DMEM | Vaughan, A., Halbert, C., Wootton, S. and Miller, A.: Lung Cancer in Mice Induced by the Jaagsiekte Sheep Retrovirus Envelope Protein is not Maintained by Rare Cancer Stem Cells, but Tumorigenicity does Correlate with Wnt Pathway Activation., Mol Cancer Res 10, 86, 2012 |
| 小鼠 | Mouse | 肺 / Lung | Collagenase Type 3: 0.1%； Deoxyribonuclease I: 0.0025% | RPMI 1640 | Hardy, C., LeMasurier, J., Belz, G., Scalzo-Inguanti, K., Yao, J., Xiang, S., Kanellakis, P., Bobik, A., Strickland, D., Rolland, J., O'Hehir, R. and Plebanski, M.: Inert 50-nm Polystyrene Nanoparticles That Modify Pulmonary Dendritic Cell Function and Inhibit Allergic Airway Inflammation., J Immunol 188, 1431, 2012 |
| 小鼠 | Mouse | 肺引流 / Lung draining | Collagenase Type 4: 0.1-0.125% | HBSS | Rayamajhi, M., Redente, E., Condon, T., Gonzalez-Juarrero, M., Riches, D. and Lenz, L.: Non-Surgical Intratracheal Instillation of Mice with Analysis of Lungs and Lung Draining Lymph Nodes by Flow Cytometry., J Vis Exp 51, 2702, 2011 |
| 小鼠 | Mouse | 樹(shù)突狀 / Dendritic | Collagenase Type 1: 0.05%； Deoxyribonuclease I: 0.002% | HBSS | Lancelin, W. and Guerrero-Plata, A.: Isolation of Mouse Lung Dendritic Cells., J Vis Exp 57, e3563, 2011 |
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