肺組織消化手冊

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Worthington組織消化指南

引言

組織消化酶主要用于組織培養和細胞生物學研究的原代細胞的分離和收集。盡管這些消化酶已經普遍應用多年，但是對其組織消化和細胞收集的作用機制卻沒有全面的理解。由此導致的結果便是選擇一種消化方法大多是隨機選擇或基于過去的經驗，而不是在*理解消化原理的基礎上選擇的消化方案，以及如何根據具體實驗目的修正消化方案以獲得更好的實驗結果。

細胞分離程序的目的是zui大程度的獲得功能活性的單個細胞，而影響結果的原因有很多，包括（不限于）以下：

1、組織類型

2、物種來源

3、動物年齡

4、遺傳改造情況（如knockouts）

5、所用的消化培養液

6、所用的酶

7、消化酶中的雜質

8、所用消化酶的工作濃度

9、溫度

10、孵育時間

通常前四個原因沒辦法選擇，而剩下的原因一般由經驗決定以獲取zui適實驗結果。

通過查閱文獻來選擇*組織消化酶和zui適消化條件的研究人員通常被互相矛盾的實驗數據搞得一頭霧水。這些巨大差異來源于組織胞外基質的復雜而動態的特性，以及過去常用的粗制、未提純的組織分離酶。另外，組織胞外基質由各種蛋白、糖蛋白、脂類和糖脂組成，這些成分的組成比例在不同物種、不同組織、甚至不同發育（年齡）之間存在明顯的差異，這些因素都極大的影響了消化方案的制定。目前通常使用的粗制酶，如鏈霉蛋白酶、NF1:250、以及含有幾種其他不同濃度蛋白酶的膠原酶、以及各種多糖酶、核酶和脂酶。

本指南總結了目前常用組織消化酶的zui適消化和細胞收集知識和方法，描述了標準的實驗步驟，以便于制定一個*的細胞分離方案，同時列舉了一些相關的參考文獻。

下表列出多種組織的適宜消化酶（含參考文獻，以組織類型和物種分類，詳見鏈接）

脂肪組織	腎上腺組織	骨組織	腦組織
軟骨組織	結腸組織	內皮組織	上皮組織
眼	心臟	腸	腎
肝	肺	淋巴結	乳腺
其它組織	其他組織	肌組織	神經組織
胰腺	腮腺	垂體	前列腺
生殖	動物鱗片	皮膚	脾臟
干細胞	胸腺	甲狀腺/甲狀旁腺	扁桃體
腫瘤

注：使用Worthington公司消化酶的文獻不限于上表鏈接中所列，一般而言，Worthington公司的組織消化酶可用于大多組織類型的消化。

肺組織消化手冊

物種	詳細來源	細胞名稱	消化用酶	培養基	參考文獻
牛	牛	Pulmonary microvessel endothelial	Collagenase Type 2: 1000 u/ml	PBS	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	豚鼠, 雄性, 250-300 克	Alveolar type II	Elastase: 40 u/ml	PBS	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
				(see reference)
豚鼠	豚鼠, 雄性, 100 克	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
人類	人類	Lung Tumor	Collagenase Type 1: 45-60 u/ml	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
			Collagenase Type 2: 15-20 u/ml
			Collagenase Type 4: 45-60 u/ml
			Elastase: 0.002%
			Deoxyribonuclease I: 0.002%
人類	人類	Alveolar epithelial	Neutral Protease: 2 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
			Trypsin: 0.25%
			Elastase: 10 u/ml
人類	人類	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
人類	人類	Lung	Neutral Protease: 2 u/ml	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
			Collagenase/Dispase: 0.1%
			Deoxyribonuclease I: 0.01%
人類	人類	Lung	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
		Fibroblasts
人類	人類, 16-24 周治療性流產	Alveolar type II	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
		Fetal
人類	人類	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
人類	人類, 12-16 周大胎兒	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
人類	人類胎兒, 80 天	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
	(包括豬胎, 70 天, 成年, 250 天)
小鼠	小鼠	Alveolar	Collagenase Type 1: 300 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
			Elastase: 4 u/ml
			Neutral Protease: 5 u/ml
			Deoxyribonuclease I: 200 u/ml
小鼠	小鼠	Pulmonary	Collagenase Type 4: 0.16%	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
			Deoxyribonuclease I: 0.1%
小鼠	小鼠	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	小鼠, 8-12 周	Lung progenitor	Collagenase/Dispase: 0.2%	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
			Neutral Protease: 5 u/ml
			Deoxyribonuclease I: 0.0025%
小鼠	小鼠	Lung tumor	Neutral Protease: 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
			Collagenase: 400 u/ml
			Deoxyribonuclease I: 50 u/ml
小鼠	小鼠	Lung	Collagenase Type 3: 0.1%	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
			Deoxyribonuclease I: 0.0025%
小鼠	小鼠	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	小鼠, 8-10 周	Lung mesenchymal stem	Collagenase Type 2: 0.2%	HBSS	Chow, K., Jun, D., Helm, K., Wagner, D. and Majka, S.: Isolation & Characterization of Hoechst(low) CD45(negative) Mouse Lung Mesenchymal Stem Cells., J Vis Exp 56, e3159, 2011
小鼠	小鼠	Dendritic	Collagenase Type 1: 0.05%	HBSS	Lancelin, W. and Guerrero-Plata, A.: Isolation of Mouse Lung Dendritic Cells., J Vis Exp 57, e3563, 2011
			Deoxyribonuclease I: 0.002%
小鼠	小鼠	Lung	Collagenase Type 1: 450 u/ml	DMEM/F12	Rock, J, Barkauskas, C, Cronc, M, Xue, Y, Harris, J, Liang, J, Noble, P and Hogan, B.: Multiple Stromal Populations Contribute to Pulmonary Fibrosis Without Evidence for Epithelial to Mesenchymal Transition., Proc Natl Acad Sci U S A 108, E1475, 2011
			Elastase: 4 u/ml
			Neutral Protease: 5 u/ml
			Deoxyribonuclease I: 0.33 u/ml
小鼠	小鼠, fe雄性, 6-12 周	Lung mononuclear	Collagenase Type 4: 500 u/ml	HBSS	Breslow, R., Rao, J., Xing, W., Hong, D., Barrett, N. and Katz, H.: Inhibition of Th2 Adaptive Immune Responses and Pulmonary Inflammation by Leukocyte Ig-Like Receptor B4 on Dendritic Cells., J Immunol 184, 1003, 2010
			Deoxyribonuclease I: 0.002%
小鼠	小鼠	Lung	Collagenase Type 2: 300 u/ml	RPMI 1640	Ferreira, C., Antunes, F., Leonard, V., Welstead, G., Richardson, C. and Cattaneo, R.: Measles Virus Infection of Alveolar Macrophages and Dendritic Cells Precedes Spread to Lymphatic Organs in Transgenic Mice Expressing Human Signaling Lymphocytic Activation Molecule (SLAM, CD150)., J Virol 84, 3033, 2010
			Deoxyribonuclease I: 0.015%
小鼠	小鼠, 胚胎, 18天	Alveolar and fibroblast	Trypsin: 2.5%	MEM	Trotter, A., Kipp, M., Schrader, R. and Beyer, C.: Combined Application of 17Beta-Estradiol and Progesterone Enhance Vascular Endothelial Growth Factor and Surfactant Protein Expression in Cultured Embryonic Lung Cells of Mice., Int J Pediatr 2009, 170491, 2009
			Deoxyribonuclease I: 0.2%
			Collagenase Type 1: 1250 u/ml
小鼠	小鼠	Lung	Collagenase Type 4: 0.1%	HBSS	Zhao, J., Zhao, J., Van Rooijen, N. and Perlman, S.: Evasion by Stealth: Inefficient Immune Activation Underlies Poor T Cell Response and Severe Disease in SARS-CoV-Infected Mice., PLoS Pathog 5, e1000636, 2009
			Deoxyribonuclease I: 0.01%
小鼠	小鼠	Dendritic	Collagenase Type 1: 0.5%	HBSS	Flano, E., Jewell, N., Durbin, R. and Durbin, J.: Methods Used to Study Respiratory Virus Infection.,Curr Protoc Cell Biol Vol. Chapter 26,, , Unit 26.3, 2009
小鼠	小鼠, 6-7 周	Leukocytes and Endothelial	Collagenase: 0.2%	PBS	Jungblut, M., Oeltze, K., Zehnter, I., Hasselmann, D. and Bosio, A.:Standardized Preparation of Single-Cell Suspensions from Mouse Lung Tissue Using the GentleMACS Dissociator., J Vis Exp 29, , 2009
			Deoxyribonuclease I: 40-80 u/ml
小鼠	小鼠	Lung	Collagenase Type 2: 0.2%	PBS	Jones, R., Capen, D., Cohen, K., Munn, L., Jain, R. and Duda, D.: A Protocol for Phenotypic Detection and Characterization of Vascular Cells of Different Origins in a Lung Neovascularization Model in Rodents., Nat Protoc 3, 388-97, 2008
小鼠	小鼠, 18-20 克	Lung	Collagenase Type 3: 0.17%	HBSS	Ebeling, C., Lam, T., Gordon, J., Hollenberg, M. and Vliagoftis, H.: Proteinase-Activated Receptor-2 Promotes Allergic Sensitization to an Inhaled Antigen through a TNF-Mediated Pathway., J Immunol 179, 2910, 2007
小鼠	小鼠	Lung	Collagenase Type 2: 300 u/ml	Dulbecco's PBS	Finotto, S., Eigenbrod, T., Karwot, R., Boross, I., Doganci, A., Ito, H., Nishimoto, N., Yoshizaki, K., Kishimoto, T., Rose-John, S., Galle, P. and Neurath, M.: Local Blockade of IL-6R Signaling Induces Lung CD4+ T Cell Apoptosis in a Murine Model of Asthma Via Regulatory T Cells., Int Immunol 19, 685, 2007
			Deoxyribonuclease I: 0.001%
小鼠	小鼠, 6-16 周	Mononuclear Cells	Collagenase Type 4: 0.1%	RPMI 1640	Abonia, J., Hallgren, J., Jones, T., Shi, T., Xu, Y., Koni, P., Flavell, R., Boyce, J., Austen, F. and Gurish, M.: Alpha-4 Integrins and VCAM-1, but not MAdCAM-1, are Essential for Recruitment of Mast Cell Progenitors to the Inflamed Lung., Blood 108, 1588-94, 2006
小鼠	小鼠, 8-12 周	Mononuclear cells	Collagenase Type 1: 300 u/ml	RPMI 1640 medium	Woolard MD, Hodge LM, Jones HP, Schoeb TR, and Simecka JW: The upper and lower respiratory tracts differ in their requirement of IFN-gamma and IL-4 in controlling respiratory mycoplasma infection and disease, J Immunol 172, 6875, 2004
			Deoxyribonuclease I: 50 u/ml
小鼠	小鼠, 6-10 周	Murine pulmonary endothelial	Collagenase Type 2: 0.25%	HBSS	Andonegui G., Bonder C., Green F., Mullaly S., Zbytnuik L., Raharjo E., and Kubes P.: Endothelium-derived Toll-like Receptor-4 is the Key Molecule in LPS-induced Neutrophil Sequestration into Lungs, J Clin Invest 111, 1011, 2003
小鼠	小鼠	Pulmonary T lymphocytes	Collagenase Type 3: 10 mg/lung	RPMI medium	Huaux F, Liu T, McGarry B, Ullenbruch M, and Phan SH.: Dual roles of IL-4 in lung injury and fibrosis, J Immunol 170, 2083, 2003
			Deoxyribonuclease I: 250 ug/lung
小鼠	小鼠	Alveolar epithelial cells	Neutral Protease:	DMEM	Paine R 3rd, Wilcoxen SE, Morris SB, Sartori C, Baleeiro CE, Matthay MA, and Christensen PJ: Transgenic overexpression of granulocyte macrophage-colony stimulating factor in the lung prevents hyperoxic lung injury, Am J Pathol 163, 2397, 2003
			Deoxyribonuclease I: 0.01%
小鼠	小鼠, 6-8 周	Lung and lymph node cells	Collagenase Type 2: 0.1%	RPMI 1640	Vermaelen KY, Carro-Muino I, Lambrecht BN, and Pauwels RA.: Specific migratory dendritic cells rapidly transport antigen from the airways to the thoracic lymph nodes, J Exp Med 193, 51, 2001
			Deoxyribonuclease I: 0.002%
小鼠	小鼠	Lung	Collagenase: 100 u/ml	Krebs-Henseleit Buffer	Freedman SD, Katz MH, Parker EM, Laposata M, Urman MY, and Alvarez JG.:A membrane lipid imbalance plays a role in the phenotypic expression of cystic fibrosis in cftr(-/-) mice, Proc Natl Acad Sci U S A 96, 13995, 1999
			Deoxyribonuclease I: 200 u/ml
小鼠	小鼠, 6-8 周	Lung cells	Collagenase Type 3: 150 u/ml	HBSS	Stampfli MR, Wiley RE, Neigh GS, Gajewska BU, Lei XF, Snider DP, Xing Z, Jordana M: GM-CSF transgene expression in the airway allows aerosolized ovalbumin to induce allergic sensitization in mice, J Clin Invest 102, 1704-14, 1998
小鼠	小鼠, 雌性, 18-20 克	Murine endothelial cells	Collagenase Type 1: 1 mg/ml	DMEM	Dong QG, Bernasconi S, Lostaglio S, De Calmanovici RW, Martin-Padura I, Breviario F, Garlanda C, Ramponi S, Mantovani A,and Vecchi A.: A general strategy for isolation of endothelial cells from murine tissues. Characterization of two endothelial cell lines from the murine lung and subcutaneous sponge implants, Arterioscler Thromb Vasc Biol 17, 1599, 1997
小鼠	小鼠, 雌性	Antigen presenting cells	Collagenase: 150 u/ml	RPMI 1640	Hamilton-Easton A, and Eichelberger M: Virus-specific antigen presentation by different subsets of cells from lung and mediastinal lymph node tissues of influenza virus-infected mice, J Virol 69, 6359, 1995
			Deoxyribonuclease I: 30 u/ml
豬	豬胎(70 天), 成年 250 天	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
	(包括人類胎兒, 80 天)
兔	兔, 新西蘭, 白色	Alveolar type II	Trypsin: 0.05%	HBSS	Scott, J.: The Role of Sera, Growth Factors, and Hormones in the In VitroProduction of Disaturated Phosphatidylcholine and Propagation of Undifferentiated Type II Alveolar Cells from the Fetal Rabbit Lung, Exp Lung Res 12, 181, 1987
兔	兔, 新西蘭, 成年的,雄性	Alveolar type II	Trypsin: 0.0025%	Joklik's MEM	Finkelstein, J., Maniscalco, W., and Shapiro, D.: Properties of Freshly Isolated Type II Alveolar Epithelial Cells, Biochim Biophys Acta 762, 398, 1983
兔	兔,新西蘭, 雄性, 2-3 公斤	Clara cells	Protease: 0.1%	HEPES	Devereux, T., and Fouts, J.: Isolation and Identification of Clara Cells From Rabbit Lung, In Vitro 16 (11), 958, 1980
兔	兔, 新西蘭, 白色, 雄性, 1.7 公斤	Lung	Pronase: 0.2%	Kreb's serum substitute solution, CMF	Gould, M., Clements, J., Jones, A., and Felts, J.: Dispersal of Rabbit Lung into Individual Viable Cells: A New Model for the Study of Lung Metabolism, Science 178, 1209, 1972
大鼠	大鼠	Lung	Collagenase Type 2: 0.2%	PBS	Jones, R., Capen, D., Cohen, K., Munn, L., Jain, R. and Duda, D.: A Protocol for Phenotypic Detection and Characterization of Vascular Cells of Different Origins in a Lung Neovascularization Model in Rodents., Nat Protoc 3, 388-97, 2008
大鼠	SD大鼠, 250-300克	Alveolar epithelial	Elastase: 3-4.5 u/ml	RPMI 1640	Chen J., Chen Z., Narasaraju T., Jin N., and Liu L.: Isolation of Highly Pure Alveolar Epithelial Type I and Type II Cells from Rat Lungs, Lab Invest 84, 727, 2004
大鼠	SD大鼠, 300-400 克	Pulmonary endothelial cells	Collagenase Type 2: 1000 u/ml	DMEM/F-12	King J, Hamil T, Creighton J, Wu S, Bhat P, McDonald F, and Stevens T:Structural and functional characteristics of lung macro- and microvascular endothelial cell phenotypes, Microvasc Res 67, 139, 2004
大鼠	SD大鼠, 125-150 克	Alveolar type I & II	Elastase: 2.5-8 u/ml	DMEM/F12	Liebler, J., Borok, Z., Li, X., Zhou, B., Sandoval, A., Kim, K.and Crandall, E.:Alveolar Epithelial Type I Cells Express Beta 2-Adrenergic Receptors and G-protein Receptor Kinase 2., J Histochem Cytochem 52, 759, 2004
			Collagenase Type 1: 1.0%
大鼠	SD大鼠, 200-225 克	Type II alveolar epithelial cells	Elastase: 4.2 u/ml	DMEM	Sunil VR, Connor AJ, Guo Y, Laskin JD, and Laskin DL: Activation of type II alveolar epithelial cells during acute endotoxemia, Am J Physiol Lung Cell Mol Physiol 282, L872, 2002
			Deoxyribonuclease I: 0.0001%
大鼠	SD大鼠, 雄性, 成年的	Alveolar	Elastase: 2.0 - 2.5 u/ml	DMEM/Ham's F-12	Kemp, P., Kim, K., Borok., Z., and Crandall, E.: Re-evaluating the Na⁺Conductance of Adult Rat Alveolar Type II Pneumocytes: Evidence for the Involvement of cGMP-Activated Cation Channels, J Physiol 536 (3), 693, 2001
				(see reference)
大鼠	Lewis大鼠, 雄性, 200-250克	Interstitial lung macrophages	Collagenase Type 1: 100 u/ml	RPMI-1640	Steinmuller C, Franke-Ullmann G, Lohmann-Matthes ML, Emmendorffer A:Local activation of nonspecific defense against a respiratory model infection by application of interferon-gamma: comparison between rat alveolar and interstitial lung macrophages, Am J Respir Cell Mol Biol 22, 481-90, 2000
			Deoxyribonuclease I: 50 u/ml
大鼠	大鼠, 雄性	Alveolar epithelial	Elastase: 40 u/ml	DMEM	Planus, E., Galiacy, S., Matthay, M., Laurent, V., Gavrilovic, J., Murphy, G., Clerici, C., Isabey, D., Lafuma, C., and d'Ortho, M.: Role of Collagenase in Mediating in Vitro Alveolar Epithelial Wound Repair, J Cell Sci 112, 243, 1999
大鼠	大鼠, 雄性, 200-250克	Pulmonary arterial myocytes	Collagenase: 0.15%	PBS	Bakhramov, A, Evans, M and Kozlowski, R: Differential effects of hypoxia on the intracellular Ca2+ concentration of myocytes isolated from different regions of the rat pulmonary arterial tree, Exp Physiol 83, 337-47, 1998
			Papain: 0.15%
			Elastase: 0.05%
大鼠	大鼠, 胚胎15天	Fetal alveolar epithelial type II	Trypsin: 0.1%	DMEM	Fraslon-Vanhulle, C, Bourbon, J and Batenburg, J: Culture of Fetal Alveolar Epithelial Type II Cells,Cell & Tissue Culture: Laboratory Procedures Vol. 2,Doyle, A., Griffiths, J., and Newell, D., John Wiley and Sons, Ltd., 13A:2.1, 1995
大鼠	SD大鼠, 雄性, 125-175 克	Alveolar epithelial	Elastase: 2 u/ml	EBSS	Kim, K., Suh, D., Lubman, R., Danto, S., Borok, Z., and Crandall, E.: Studies on the Mechanisms of Active Ion Fluxes Across Alveolar Epithelial Cell Monolayers, J Tiss Cul Meth 14, 187, 1992
大鼠	Wistar大鼠	Epithelial	Trypsin: 0.1%	HBSS	Jassal, D., Han, R., Caniggia, I., Post, M., and Tanswell, A.: Growth of Distal Fetal Rat Lung Epithelial Cells in a Defined Serum-Free Medium, In Vitro Cell Dev Biol 27A, 625, 1991
大鼠	SD大鼠, 8 天	Interstitial	Trypsin: 1.125%	HEPES buffer	Berk, J., Franzblau, C., and Goldstein, R.: Recombinant Interleukin-1beta Inhibits Elastin Formation by a Neonatal Rat Lung Fibroblast Subtype, J Biol Chem266, 3192, 1991
大鼠	Wistar大鼠, 雌性, vir克in	Alveolar epithelial type II	Trypsin: 1%	Eagle's MEM	Fraslon, C., Rolland, G., Bourbon, J., Rieutort, M., and Valenza, C.: Culture of Fetal Alveolar Epithelial Type II Cells in Serum-Free Medium, In Vitro Cell Dev Biol27A, 843, 1991
大鼠	大鼠,食魚動物 344, 雄性	Alveolar type II pneumocytes	Elastase: 40 u/ml	HEPES buffer	Mangum, J., Everitt, J., Bonner, J., Moore, L., and Brody, A.: Co-Culture of Primary Pulmonary Cells to Model Alveolar Injury and Translocation of Proteins, In Vitro Cell Dev Biol 26, 1135, 1990
				(see reference)
大鼠	SD大鼠, 雄性, 250-400 克	Alveolar type II	Elastase: 40 u/ml	Phosphate-buffered medium	Ma, J., LaCagnin, L., Bowman, L., and Miles, P.: Carbon Tetrachloride Inhibits Synthesis of Pulmonary Surfactant Disaturated Phosphatidylcholines and ATP Production in Alveolar Type II Cells, Biochim Biophys Acta 1003, 136, 1989
				(see reference)
大鼠	Wistar大鼠, 成年的, 雄性和懷孕母鼠 (已知妊娠)	Alveolar type II	Trypsin: 0.1%	RPMI 1640	Batenburg, J., Otto-Verberne, C., Have-Opbroek, A., and Klazinga, W.:Isolation of Alveolar Type II Cells from Fetal Rat Lung by Differential Adherence in Monolayer Culture, Biochim Biophys Acta 960, 441, 1988
大鼠	SD大鼠, 雄性, 150 - 200 克	Pneumocytes type II	Trypsin: 0.30%	BSS	Brown, L. and Longmore, W.: Altered Phospholipid Secretion in Type II Pneumocytes Isolated from Streptozotocin-diabetic Rats, Biochim Biophys Acta878, 258, 1986
大鼠	SD大鼠, 雄性, 180-200 克	Alveolar type II	Elastase: 4.3 u/ml	HEPES	Dobbs, L., Gonzalez R., and Williams, M.: An Improved Method for Isolating Type ll Cells in High Yield and Purity, Am Rev Respir Dis 134, 141, 1986
				(see reference)
大鼠	SD大鼠, 雄性	Alveolar type I	Trypsin: 0.05%	DMEM	Weller, N., and Karnovksy, M.: Isolation of Pulmonary Alveolar Type I Cells From Adult Rats, Am J Pathol 124, 448, 1986
大鼠	大鼠, 胎兒, 19 天	Fibroblasts & type II	Trypsin: 0.05%	HBSS: serum free MEM, CMF	Post, M., Torday, J., and Smith, B.: Alveolar Type II Cells Isolated from Fetal Rat Lung Organotypic Cultures Synthesize and Secrete Surfactant-Associated Phospholipids and Respond to Fibroblast-Pneumonocyte Factor, Exp Lung Res 7, 53, 1984
大鼠	SD大鼠, 雄性, 150-400 克	Alveolar type II	Elastase: 4 u/ml	Auto-Pow Eagle's modified MEM	Goodman, B., Fleischer, R., and Crandall, E.: Evidence for Active Na+ Transport by Cultured Monolayers of Pulmonary Alveolar Epithelial Cells, Am J Physiol 245, C78, 1983
大鼠	SD大鼠, 雄性/雌性, 180-250 克	Alveolar type II	Trypsin: 0.30%	BSS	Mason, R., Williams, M., Greenleaf, R., and Clements, J.: Isolation and Properties of Type II Alveolar Cells from Rat Lung, Am Rev Respir Dis 115, 1015, 1977
大鼠	Wistar大鼠, 無病原體	Alveolar type II	Trypsin: 0.50%	Earle's MEM	King, R.: Metabolic Fate of the Apoproteins of Pulmonary Surfactant, Am Rev Respir Dis 115, 73, 1977
大鼠	大鼠, 胎兒, 19 天	Alveolar pneumonocytes, type II	Trypsin: 0.1 %	HBSS, CMF	Douglas, W., and Teel, R.: An Organotypic in Vitro Model System for Studying Pulmonary Surfactant Production by Type II Alveolar Pneumonocytes, Am Rev Respir Dis 113, 17, 1976
大鼠	大鼠, 成年的	Lung	Collagenase: 0.1%	Moscona saline, CMF	Douglas, W., and Kaighn, M.: Clonal Isolation of Differentiated Rat Lung Cells,In Vitro 10, 230, 1974
大鼠	SD大鼠, 雄性, 100 克	Alveolar type II	Trypsin: 1.0%	Joklik's medium	Kikkawa, Y., and Yoneda, K.: Type II Epithelial Cell of the Lung. I. Method of Isolation, Lab Invest 30, 76, 1974

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