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内皮细胞组织消化手册

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Worthington组织消化指南

引言

组织消化酶主要用于组织培养和细胞生物学研究的原代细胞的分离和收集。尽管这些消化酶已经普遍应用多年,但是对其组织消化和细胞收集的作用机制却没有全面的理解。  由此导致的结果便是选择一种消化方法大多是随机选择或基于过去的经验,而不是在*理解消化原理的基础上选择的消化方案,以及如何根据具体实验目的修正消化方案以获得更好的实验结果。

细胞分离程序的目的是zui大程度的获得功能活性的单个细胞,而影响结果的原因有很多,包括(不限于)以下:

1、组织类型

2、物种来源

3、动物年龄

4、遗传改造情况(如knockouts

5、所用的消化培养液

6、所用的酶

7、消化酶中的杂质

8、所用消化酶的工作浓度

9、温度

10孵育时间

通常前四个原因没办法选择,而剩下的原因一般由经验决定以获取zui适实验结果。

通过查阅文献来选择*组织消化酶和zui适消化条件的研究人员通常被互相矛盾的实验数据搞得一头雾水。这些巨大差异来源于组织胞外基质的复杂而动态的特性,以及过去常用的粗制、未提纯的组织分离酶。另外,组织胞外基质由各种蛋白、糖蛋白、脂类和糖脂组成,这些成分的组成比例在不同物种、不同组织、甚至不同发育(年龄)之间存在明显的差异,这些因素都极大的影响了消化方案的制定。目前通常使用的粗制酶,如链霉蛋白酶、NF1:250、以及含有几种其他不同浓度蛋白酶的胶原酶、以及各种多糖酶、核酶和脂酶。

本指南总结了目前常用组织消化酶的zui适消化和细胞收集知识和方法,描述了标准的实验步骤, 便于制定一个*的细胞分离方案,同时列举了一些相关的参考文献。

下表列出多种组织的适宜消化酶(含参考文献,以组织类型和物种分类,详见链接)

脂肪组织

肾上腺组织

骨组织

脑组织

软骨组织

结肠组织

内皮组织

上皮组织

心脏

淋巴结

乳腺

其它组织

其他组织

肌组织

神经组织

胰腺

腮腺

垂体

前列腺

生殖

动物鳞片

皮肤

脾脏

干细胞

胸腺

甲状腺/甲状旁腺

扁桃体

肿瘤

 

 

 

注:使用Worthington公司消化酶的文献不限于上表链接中所列,一般而言,Worthington公司的组织消化酶可用于大多组织类型的消化。

 

内皮细胞组织消化手册

 

物种详细来源细胞名称消化用酶培养基参考文献
Bovine umbilical cord (BUVEC)Collagenase: 0.1%Dulbecco's/Ham F-12Ricken Albert M, Traenkner Anja, Merkwitz Claudia, Hummitzsch Katja, Grosche Jens, Spanel-Borowski Katharina: The short prolactin receptor predominates in endothelial cells of micro- and macrovascular origin, J Vasc Res 44, 19-30, 2007
Pulmonary artery endothelial and smooth muscle cellsCollagenase: 0.04-0.05%RPMI-1640Yu, M., McAndrew, R., Al-Saghir, R., Maier, K., Medhora, M., Roman, R. and Jacobs, E.: Nitric Oxide Contributes to 20-HETE-Induced Relaxation of Pulmonary Arteries., J Appl Physiol 93, 1391, 2002
Soybean Trypsin Inhibitor: 0.04%
牛, (欧洲牛), 牛犊EndothelialCollagenase: 1000 u/mlPBS, CMFDel Vecchio, P.J., Siflinger-Birnboim, A., Belloni, P.N., Holleron, L.A., Lum, H., and Malik, A.B.: Culture and Characterization of Pulmonary Microvascular Endothelial Cell, In Vitro Cell Dev Biol 28A, 711, 1992
Pulmonary
牛犊EndothelialTrypsin: 0.25%HEPESVender, R.: Role of Endothelial Cells in the Proliferative Response of Cultured Pulmonary Vascular Smooth Muscle Cells to Reduced Oxygen Tension, In Vitro Cell Dev Biol 28A, 403, 1992
牛, 成年的Cerebral arteryCollagenase: 0.2%HBSSMachi, T., Kassell, N.F., and Scheld, W.M.: Isolation and Characterization of Endothelial Cells From Bovine Cerebral Arteries, In Vitro Cell Dev Biol 26, 291, 1990
Endothelial
EndothelialTrypsin: 0.05%Krebs Ringer solutionDeNucci, G., Gryglewski, R.J., Warner, T.D., and Vane, J.R.: Receptor-Mediated Release of Endothelium-Derived Relaxing Factor and Prostacyclin From Bovine Aortic Endothelial Cells Is Coupled, Proc Natl Acad Sci U S A 85, 2334, 1988
Aortic
Endothelial, pulmonary arteryCollagenase: 0.1%CMF-Dulbecco's PBSMartin, T.: Formation of Diacylglycerol by a Phospholipase D-phosphatidate Phosphatase Pathway Specific for Phosphatidylcholine in Endothelial Cells, Biochim Biophys Acta 962, 282, 1988
EndothelialCollagenase Type 2: 0.1%PBSCarson, M.P. and Haudenschild, C.C.: Microvascular Endothelium and Pericytes: High Yield, Low Passage Cultures, In Vitro Cell Dev Biol 22, 344, 1986
Aorta
EndothelialCollagenase Type 2: 0.1%DMEMKinsella, Michael G. and Wight, Thomas N.: Modulation of Sulfated Proteoglycan Synthesis by Bovine Aortic Endothelial Cells During Migration, J Cell Biol 102, 679, 1986
Aortic
EndothelialCollagenase: 0.5%DMEM/Ham's F-12Gospodarowicz, D., Massoglia, S., Cheng, J., and Fujii, D.: Effect of Fibroblast Growth Factor and Lipoproteins on the Proliferation of Endothelial Cells Derived From Bovine Adrenal Cortex, Brain Cortex, and Corpus Luteum Capillaries, J Cell Physiol 127, 121, 1986
EndothelialCollagenase Type 2: 0.2%Dulbecco's PBSGoetz, I.., Warren, J., Estrada, C., Roberts, E., and Krause, D.: Long-Term C*tion of Arterial and Capillary Endothelium From Adult Bovine Brain, In Vitro Cell Dev Biol 21, 172, 1985
Brain arteries
牛犊EndothelialCollagenase: 0.75%DMEMVoyta, J., Via, D., Butterfield, C., and Zetter, B.: Identification and Isolation of Endothelial Cells Based on Their Increased Uptake of Acetylated-Low Density Lipoprotein, J Cell Biol 99, 2034, 1984
Smooth muscle
EndothelialTrypsin: 0.2%PBS: DMEMScott, D., Murray, J., and Barnes, M.: Investigation of the Attachment of Bovine Corneal Endothelial Cells, Exp Cell Res 144, 472, 1983
Corneal
牛, 2-3 周EndothelialCollagenase Type 1: 0.2%RPMI 1640 w/1% Fetal Bovine SerumLee, S., Douglas, W., Deneke, S., and Fanburg, B.: Ultrastructural Changes in Bovine Pulmonary Artery Endothelial Cells Exposed to 80% O2 In Vitro, In Vitro 19, 714, 1983
Pulmonary artery
Endothelial, CornealTrypsin: 0.05%0.01M Phosphate buffer with 0.02% EDTARobinson, J. and Gospodarowicz, D.: Glycosaminoglycans Synthesized by Cultured Bovine Corneal Endothelial Cells, J Cell Physiol 117, 368, 1983
0.9% NaCl (See Reference)
EndothelialCollagenase Type 1: 0.10%PBSOlander, J., Marasa, J., Kimes, R., Johnston, G., and Feder, J.: An assay measuring the stimulation of several types of bovine endothelial cells by growth factor(s) derived from cultured human tumor cells, In Vitro 18, 99, 1982
Subclavian vein
AorticCollagenase Type 2: 0.10%PBSMakarski, J. S.: Stimulation of cyclic AMP production by vasoactive agents in cultured bovine aortic and pulmonary artery endothelial cells, In Vitro 17, 450, 1981
Pulmonary artery
牛犊, 胎儿, 4-9 个月EndothelialCollagenase Type 1: 0.25%PBSRosen, E., Mueller, S., Noveral, J., and Levine, E.: Proliferative Characteristics of Clonal Endothelial Cell Strains, J Cell Physiol 107, 123, 1981
牛, 成年的AortaCollagenase Type 1: 125 u/mlDulbecco's PBS with calcium and magnesiumCotta-Pereira, G., Sage, H., Bornstein, P., Ross, R., and Schwartz, S.: Studies of Morphologically Atypical ("Sprouting") Cultures of Bovine Aortic Endothelial Cells. Growth Characteristics and Connective Tissue Protein Synthesis, J Cell Physiol 102, 183, 1980
牛, 年幼的Pulmonary arteryCollagenase: 0.1%Medium 199Ryan, U., Mortara, M., and Whitaker, C.: Methods for Microcarrier Culture of Bovine Pulmonary Artery Endothelial Cells Avoiding the Use of Enzymes, Tissue Cell12, 619, 1980
ForeskinCollagenase: 0.5%Dulbecco's MEM w/10% calf serumFolkman, J., Haudenschild, C. C., and Zetter, B. R.: Long-term Culture of Capillary Endothlial Cells, Proc Natl Acad Sci U S A 76, 5217, 1979
Saphenous VeinCollagenase: 0.01%PBSEskin, S., Sybers, H., Trevino, L., Lie, J., and Chimoskey, J.: Comparison of Tissue-Cultured Bovine Endothelial Cells from Aorta and Saphenous Vein, In Vitro 14, 903, 1978
Aorta
EndothelialCollagenase Type 2: 0.1%PBSSchwartz, S.M.: Selection and Characterization of Bovine Aortic Endothelial Cells,In Vitro 14, 966, 1978
Thoracic aorta 
Saphenous veins
Pulmonary arteryCollagenase Type 2: 0.25%Puck's solutionRyan, U.S., Clements, E., Habliston, D., and Ryan, J.W.: Isolation And Culture of Pulmonary Artery Endothelial Cells, Tissue Cell 10, 535, 1978
牛犊EndothelialCollagenase Type 2: 0.1%DMEMHoward, B., Macarak, E., Gunson, D., and Kefalides, N.: Characterization of the Collagen Synthesized by Endothelial Cells in Culture, Proc Natl Acad Sci U S A 73 (7), 2361, 1976
MicrovesselsCollagenase Type 4: 0.1%DMEMGerhart, D. Z., Broderius, M. A., and Drewes, L. R.: Cutlured Human and Canine Endothelial Cells from Brain Microvessels, Brain Res Bull 21, 785, 1988
(包括人类)
狗, 杂种狗, 成年的EndotheliumTrypsin: 0.1%Earle's PBS, CMFFord, J., Burkel, W., and Kahn, R.: Isolation of Adult Canine Venous Endothelium for Tissue Culture, In Vitro 17, 44, 1981
Jugular vein
豚鼠豚鼠, 雌性, 300-350克Coronary endothelialCollagenase Type 2: 0.1%see referenceBuxton I L, Kaiser R A, Oxhorn B C, Cheek D J: Evidence supporting the Nucleotide Axis Hypothesis: ATP release and metabolism by coronary endothelium,Am J Physiol Heart Circ Physiol 281, H1657-66, 2001
人类人类Vascular endothelialCollagenase Type 1: 0.1%HBSSGanguly, A., Zhang, H., Sharma, R., Parsons, S. and Pa, K.: Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions., J Vis Exp 66, e4032, 2012
人类人类, 18-68 yrCorneal endothelialCollagenase: 0.2%DMEM/F12Li Wei, Sabater Alfonso L, Chen Ying-Ting, Hayashida Yasutaka, Chen Szu-Yu, He Hua, Tseng Scheffer C G: A novel method of isolation, preservation, and expansion of human corneal endothelial cells, Inv Ophthal Visual Sci 48, 614-20, 2007
Neutral Protease: 1.0%
人类人类Endothelial and vascular smooth muscleCollagenase Type 1: 0.2%HBSSMoss, S., Bates, M., Parrino, P. and Woods, TC.: Isolation of Endothelial Cells and Vascular Smooth Muscle Cells from Internal Mammary Artery Tissue., Ochsner J 7, 133, 2007
人类人类HUVECCollagenase Type 1: 0.1%HBSSDavis, J., Crampton, S. and Hughes. C.: Isolation of Human Umbilical Vein Endothelial Cells (HUVEC)., J Vis Exp 3, 183, 2007
人类人类HUVECCollagenase Type 4: 0.1%RPMI 1640Silva AP, Kaufmann JE, Vivancos C, Fakan S, Cavadas C, Shaw P, Brunner HR, Vischer U, and Grouzmann E.: Neuropeptide Y expression, localization and cellular transducing effects in HUVEC, Biol Cell 97(6), 457, 2005
人类人类EndothelialCollagenase Type 2: 0.1%DMEMPa, V., Logan, A., Watkinson, J., Uz-Zaman, S., Sheppard, M., Ramsden, J. and Eggo, M.: Isolation and Characterization of Human Thyroid Endothelial Cells.,Am J Physiol Endocrinol Metab 284, E168, 2003
人类人类Esophageal microvascular endothelialCollagenase Type 2: 0.2%MCDB-131Rafiee, P., Ogawa, H., Heidemann, J., Li, M., Aslam, M., Lamirand, T., Fisher, P., Graewin, S., Dwinell, M., Johnson, C., Shaker, R. and Binion, D.: Isolation and Characterization of Human Esophageal Microvascular Endothelial Cells: Mechanisms of Inflammatory Activation., Am J Physiol Gastrointest Liver Physiol285, G1277, 2003
人类人类Colonic endothelial cellsCollagenase Type 2: 0.25%HBSS/5%FBSWang D., Lehman R., Donner D., Matli M., Warren R., and Welton M.: Expression and Endocytosis of VEGF and Its Receptors in Human Colonic Vascular Endothelial Cells, Am J Physiol/Gastro 282, G1088, 2002
人类人类Pulmonary vascular endothelial cellsNeutral Protease: 1.18 u/mlM199Muller AM, Hermanns MI, Skrzynski C, Nesslinger M, Muller KM, and Kirkpatrick CJ.: Expression of the endothelial markers PECAM-1, vWf, and CD34 in vivo and in vitro, Exp Mol Pathol 72, 221, 2002
Elastase: 10 u/ml
人类人类HUVECCollagenase Type 2: 0.1%PBSTakano Manabu, Meneshian Avedis, Sheikh Emran, Yamakawa Yasuhiko, Wilkins Kirsten Bass, Hopkins Elise A, Bulkley Gregory B: Rapid upregulation of endothelial P-selectin expression via reactive oxygen species generation, Am J Physiol Heart Circ Physiol 283, H2054-61, 2002
人类人类EndothelialTrypsin: 2%PBSGoolcharran, C., Cleland, J., Keck, R., Jones, A., and Borchardt, R.: Comparsion of the Rates of Deamidation, Diketopiperazine Formation and Oxidation in Recombinant Human Vascular Endothelial Growth Factor and Model Peptides,AAPS PharmSci 2 (1), 5, 2000
人类人类HUVEC, porcine pulmonary arterial endothelial cellsCollagenase Type 2: 0.2%DMEMKwak HJ, Lee SJ, Lee YH, Ryu CH, Koh KN, Choi HY, and Koh GY.: Angiopoietin-1 inhibits irradiation- and mannitol-induced apoptosis in endothelial cells, Circulation 101(19), 2317, 2000
人类人类Vascular endothelial cellsCollagenase: 0.1%DMEMSchonbeck U, Sukhova GK, Graber P, Coulter S, Libby P: Augmented expression of cyclooxygenase-2 in human atherosclerotic lesions, Am J Pathol 155, 1281-91, 1999
人类人类Hepatic endothelial cellsCollagenase: 0.2%DMEMSanyal AJ, and Mirshahi F.: A simplified method for the isolation and culture of endothelial cells from pseudointima of transjugular intrahepatic portasystemic shunts, Lab Invest 78(11), 1469, 1998
人类人类Foreskin microvascular endothelialTrypsin: 0.3%HBSSWojta J, Gallicchio M, Zoellner H, Filonzi E, Hamilton J, McGrath K: Interleukin-4 stimulates expression of urokinase-type-plasminogen activator in cultured human foreskin microvascular endothelial cells, Blood 81, 3285-92, 1993
人类人类EndothelialTrypsin: 0.3.%HBSSLee, K, Lawley, T, Xu, Y, and Swerlick, R.: VCAM-1-, ELAM-1-, and ICAM-1-Independent Adhesion of Melanoma Cells to Cultured Human Dermal Microvascular Endothelial Cells, J Invest Dermatol 98, 79, 1992
(see reference)
人类人类Vascular endothelialNeutral Protease: 0.15%M-199Farber HW, Antonov AS, Romanov YA, Smirnov VN, Scarfo LM, Beer DJ: Cytokine secretion by human aortic endothelial cells is related to degree of atherosclerosis,Am J Physiol 262, H1088-95, 1992
Trypsin: 0.25%
人类人类Umbilical veinCollagenase: 0.1%Cord BufferGrant, D.S., Lelkes, P.I., Fukuda, K., Kleinman, H.K.: Intracellular Mechanisms Involved in Basement Membrane Induced Blood Vessel Differentiation In Vitro, In Vitro Cell Dev Biol 27, 327, 1991
HUVEC(See Reference)
人类人类Crypt cellsCollagenase: 125 u/mlRMPI 1640Whitehead, R., and Eeden, P.: A Method For the Prolonged Culture of Colonic Epithelial Cells, J Tiss Cul Meth 13, 103, 1991
人类人类EndothelialCollagenase Type 1: 0.2%Medium 199Fischer, E., Stingl, A., and Kirkpatrick, C.: Migration Assay for Endothelial Cells in Multiwells Application to Studies on the Effect of Opioids, J Immunol Methods 128, 235, 1990
人类人类Human umbilical vein endothelial cellsCollagenase Type 2: 75 u/mlM199Muller WA, Ratti CM, McDonnell SL, Cohn ZA: A human endothelial cell-restricted, externally disposed plasmalemmal protein enriched in intercellular junctions, J Exp Med 170, 399-414, 1989
人类人类MicrovesselsCollagenase Type 4: 0.1%DMEMGerhart, D. Z., Broderius, M. A., and Drewes, L. R.: Cutlured Human and Canine Endothelial Cells from Brain Microvessels, Brain Res Bull 21, 785, 1988
人类人类Endothelial/HUVECTrypsin: 0.3%HBSS/PBS, Medium 199Kubota, Y., Kleinman, H., Martin, G., and Lawley, T.: Role of Laminin and Basement Membrane in Morphological Differentiation of Human Endothelial Cells into Capillary-like Structures, J Cell Biol 107, 1589, 1988
Foreskin & umbilical cord(see reference)
人类人类Umbilical cordCollagenase: 0.1%HEPESHoshi, H., Kan, M., Chen, J., and McKeehan, W.: Comparative Endocrinology-Paracrinology-Autocrinology of Human Adult Large Vessel Endothelial and Smooth Muscle Cells, In Vitro Cell Dev Biol 24 (4), 309, 1988
Smooth muscle
人类人类EndothelialCollagenase Type 2: 0.1%PBS, CMFSharefkin, J.B., Fairchild, K.D., Albus, R.A., Cruess, D.F., and Rich, N.M.: The Cytoxic Effect of Surgical Glove Powder Particles on Adult Human Vascular Endothelial Cell Cultures: Implications for Clinical Uses of Tissue Culture Techniques, J Surg Res 41, 463, 1986
Saphenous vein
人类人类EndothelialCollagenase: 0.1%HEPESHoshi, H., and McKeehan, W.: Isolation, Growth Requirements, Cloning, Prostacylclin Production and Life-Span of Human Adult Endothelial Cells in Low Serum Culture Medium, In Vitro Cell Dev Biol 22 (1), 51, 1986
人类人类EndothelialTrypsin: 0.3%PBSMarks, R.M., Czerniecki, M., and Penny, R.: Human Dermal Microvascular Endothelial Cells: An Improved Method for Tissue Culture and Description of Some Singular Properties in Culture, In Vitro Cell Dev Biol 21, 627, 1985
Dermal
人类人类FibroblastsHyaluronidase: 0.10%DMEMGordon, P., Sussman, I., and Hatcher, V.: Long-Term Culture of Human Endothelial Cells, In Vitro 19, 661, 1983
Foreskin
人类人类Iliac arteriesCollagenase: 0.25%PBS w/Ca++, Mg++, & BSAGlassberg, M., Bern, M., Coughlin, S., Haudenschild, C., Hoyer, L., and Antoniades, H.: Cultured Endothelial Cells Derived from the Human Iliac Arteries,In Vitro 18, 859, 1982
(see reference)
人类人类, 成年的Peripheral blood mononuclearCollagenase: 0.25%RPMI 1640Ashida, E., Johnson, A., and Lipsky, P.: Human endothelial cell-lymphocyte interaction. Endothelial cells function as accessory cells necessary for mitogen-induced human T lymphocyte activation in vitro, J Clin Invest 67, 1490, 1981
Monocytes
T cells
Endothelial
人类人类Microvascular endothelialNeutral Protease: at 1000 u/mlKonigsberg's modification of HBSSSherer, G., Fitzharris, T., Faulk, W., and LeRoy, E.: C*tion of Microvascular Endothelial Cells from Human Preputial Skin, In Vitro 16, 675, 1980
Neonatal foreskins(See Reference)
人类人类 (成年人和小孩)ForeskinCollagenase: 0.5%Dulbecco's MEM w/10% calf serumFolkman, J., Haudenschild, C. C., and Zetter, B. R.: Long-term Culture of Capillary Endothlial Cells, Proc Natl Acad Sci U S A 76, 5217, 1979
人类人类Umbilical veinTrypsin: 100 礸/mlTris-HCl,0.2 MJaffe, E.A., Minick, C.R., Adelman, B., Becker, C.G., and Nachman, R.: Synthesis of Basement Membrane Collagen By Cultured Human Endothelial Cells, J Exp Med 144, 209, 1976
人类人类Umbilical veinCollagenase Type 1: 125 u/mlDulbecco's PBSGimbrone Jr., M.A.: Culture of Vascular Endothelium, Prog Hemost Thromb 3, 1, 1976
人类人类Umbilical cordCollagenase: 0.1%Dulbecco's PBSGimbrone, M.A., Cotran, R.S., and Folkman, J.: Human Vascular Endothelial Cells in Culture: Growth and DNA Synthesis, J Cell Biol 60, 673, 1974
人类人类Umbilical cordCollagenase: 0.2%Cord bufferJaffe, E., Nachman, R., Becker, C., and Minick, C.: Culture of Human Endothelial Cells Derived from Umbilical Veins. Identification by Morphologic and Immunologic Criteria, J Clin Invest 52, 2745, 1973
(See Reference)
人类人类Umbilical cordTrypsin NF 1:250: 0.125%Saline, normalLewis, L.J., Haok, J.C., Maca, R.D., and Fry, G.L.: Replication of Human Endothelial Cells in Culture, Science 181, 452, 1973
人类人类Umbilical cordTrypsin NF 1:250: 0.25%CMF solutionFryer, D.G., Birnbaum, G., and Luttrell, C.N.: Human Endothelium in Cell Culture, J Atheroscler Res 6, 151, 1966
小鼠小鼠Endothelial lungCollagenase Type 1: 0.2%DMEMWang, J., Sun, C., Gerdes, N., Liu, C., Liao, M., Liu, J., Shi, M., He, A., Zhou, Y., Sukhova, G., Chen, H., Cheng, X., Kuzuya, M., Murohara, T., Zhang, J., Cheng, X., Jiang, M., Shull, G. and Rogers, S.: Interleukin 18 Function in Atherosclerosis is Mediated by the Interleukin 18 Receptor and the Na-Cl Co-Transporter., Nat Med 21, 820-6, 2015
小鼠小鼠Tumor endothelialCollagenase Type 1: 0.2%HBSSKazerounian, S., Gerald, D., Huang, M., Chin, R, Udayakumar, D, Zheng, N., O'Donnell, R., Perruzzi, C., Mangiante, L., Pourat, J., Phung, T., Bravo-Nuevo, A., Shechter, S., McNamara, S. and Duhadaway, K.: RhoB Differentially Controls Akt Function in Tumor Cells and Stromal Endothelial Cells During Breast Tumorigenesis., Cancer Res 73, 50, 2013
小鼠小鼠, 8-14 weekEndothelialCollagenase Type 4: 0.2%HBSSImoukhuede, P. and Popel, A.: Expression of VEGF Receptors on Endothelial Cells in Mouse Skeletal Muscle., PLoS ONE 7, e44791, 2012
小鼠小鼠, 7-10 周Liver endothelialCollagenase: 0.03%DMEMFollenzi, A., Benten, D., Novikoff, P., Faulkner, L., Raut, S. and Gupta, S.:Transplanted Endothelial Cells Repopulate the Liver Endothelium and Correct the Phenotype of Hemophilia A Mice., J Clin Invest 118, 935, 2008
小鼠小鼠, 4 周Endothelial kidneyCollagenase Type 1: 0.1%DMEMKondo, S., Scheef, E., Sheibani, N. and Sorenson, C.: PECAM-1 Isoform-Specific Regulation of Kidney Endothelial Cell Migration and Capillary Morphogenesis., Am J Physiol Cell Physiol 292, C2070, 2007
小鼠小鼠, 胚胎或卵黄囊EndothelialCollagenase Type 3: 200 u/mlPBSBraren Rickmer, Hu Huiqing, Kim YungHae, Beggs HilaryE, Reichardt LouisF, Wang Rong: Endothelial FAK is essential for vascular network stability, cell survival, and lamellipodial formation, J Cell Biol 172, 151-62, 2006
Deoxyribonuclease I: 0.001%
小鼠小鼠, 初生的Microvascular endothelialNeutral Protease: 0.005%DMEMCha, S., Talavera, D., Demir, E., Nath, A. and Sierra-Honigmann, M.: A Method of Isolation and Culture of Microvascular Endothelial Cells from Mouse Skin.,Microvasc Res 70, 198, 2005
Collagenase Type 1: 4%
小鼠小鼠, 4 周Retinal endothelial cellsCollagenase Type 1: 0.1%DMEMSu X, Sorenson CM, and Sheibani N.: Isolation and characterization of murine retinal endothelial cells, Mol Vis 9, 171, 2003
小鼠小鼠, 雄性Endothelial cells from lymph nodeCollagenase Type 1: 0.1%PBSIzawa D, Tanaka T, Saito K, Ogihara H, Usui T, Kawamoto S, Matsubara K, Okubo K, and Miyasaka M: Expression profile of active genes in mouse lymph node high endothelial cells, Int Immunol 11(12), 1989, 1999
小鼠小鼠, 2-4 周Cerebrovascular EndothelialCollagenase/Dispase: 0.1%PBSSapatino, B., Welsh, C., Smith, C., Bebo, B., and Linticum, D.: Cloned Mouse Cerebrovascular Endothelial Cells That Maintain Their Differentiation Markers for Factor VIII, Low Density Lipoprotein, and Angiotensin-Converting Enzyme, In Vitro Cell Dev Biol 29A, 923, 1993
猪, 6 个月Valvular endothelialNeutral Protease: 2 u/mlPBSBalaoing, L., Post, A., Lin, A., Tseng, H., Moake, J. and Grande-Allen, K.: Laminin Peptide-Immobilized Hydrogels Modulate Valve Endothelial Cell Hemostatic Regulation., PLoS ONE 10, e0130749, 2015
Collagenase Type 2: 60 u/ml
Valvular endothelialCollagenase Type 2: 300-600 u/mlDMEMGould, R. and Butcher, J.: Isolation of Valvular Endothelial Cells., J Vis Exp 46, e2158, 2010
猪 EndothelialCollagenase Type 1: DMEM w/ 10% calf serumNugent, H., and Edelman, E.: Endothelial Implants Provide Long-Term Control of Vascular Repair in a Porcine Model of Arterial Injury, J Surg Res 99, 228, 2001
(包括 牛)
HUVEC, porcine pulmonary arterial endothelial cellsCollagenase Type 2: 0.2%DMEMKwak HJ, Lee SJ, Lee YH, Ryu CH, Koh KN, Choi HY, and Koh GY.: Angiopoietin-1 inhibits irradiation- and mannitol-induced apoptosis in endothelial cells, Circulation 101(19), 2317, 2000
猪, 6-7 个月Porcine pulmonary endothelialCollagenase Type 1: 0.3%RPMI 1640Hill-Kapturczak N, Kapturczak MH, Block ER, Pa JM, Malinski T, Madsen KM, and Tisher CC.: Angiotensin II-stimulated nitric oxide release from porcine pulmonary endothelium is mediated by angiotensin IV, J Am Soc Nephrol 10(3), 481, 1999
EndothelialTrypsin: 0.25%Medium 199Shasby, S.: Endothelial Cells Grown On Permeable Membrane Supports, J Tiss Cul Meth 14, 247, 1992
EndothelialTrypsin: 0.05%DMEMVischer, P., and Buddecke, E.: Alteration of Glycosytransferase Activities during Proliferation of C*ted Arterial Endothelial Cells and Smooth Muscle Cells, Exp Cell Res 158, 15, 1985
EndothelialCollagenase Type 2: 0.1%Dulbecco-Vogt MEM w/o serumDickinson, E. and Slakey, L.: Plasma-derived Serum as a Selective Agent to Obtain Endothelial Cultures from Swine Aorta, In Vitro 18, 63, 1982
Aortic
猪, 30-40 公斤EndotheliaCollagenase Type 4: 0.025%Medium 199Merrilees, M.J., and Scott, L.: Interaction of Aortic Endothelial and Smooth Muscle Cells in Culture, Atherosclerosis 39, 147, 1981
Aortic
猪, 20-30 周EndothelialCollagenase: 0.1%Medium 199 w/BSS and HEPES or NaHCO3Slater, D.N., and Sloan, J.M.: The Porcine Endothelial Cell in Tissue Culture,Atherosclerosis 21, 259, 1975
Aortas 
Veins
猪, 60-100 天AortaTrypsin: 0.1%Phosphate bufferCoulson, W.F.: The Effect Of Proteolytic Enzymes on the Tensile Strength of Whole Aorta and Isolated Aortic Elastin, Biochim Biophys Acta 237, 378, 1971
(see reference)
Corneal endothelial cells (CEC)Hyaluronidase: 0.05%DMEMChoi, J., Ko, M., and Kay, E.: Subcellular Localization of the Expressed 18 kDa FGF-2 Isoform in Corneal Endothelial Cells, Mol Vis 6, 222, 2000
兔, 2-3公斤Endothelial, aorticElastase: 0.2%Hanks solutionHaley, N., Shio, H., Fowler, S.: Characterization of lipid-laden aortic cells from cholesterol-fed rabbits. I. Resolution of aortic cell populations by metrizamide density gradient centrifugation, Lab Invest 37, 287, 1977
大鼠大鼠, SD, 雄性, 250-300 克Smooth muscle, aortaSoybean Trypsin Inhibitor: 0.25%HBSS with 0.2 mM Ca++Schwertschlag, U.S., and Whorton, A.R.: Plaet-Activating Factor-Induced Homologous and Heterologous Desensitization In Cultured Vascular Smooth Muscle Cells, J Biol Chem 263, 13791, 1988
大鼠大鼠, SD, 雄性, 350 - 450 gLipocytesCollagenase: 0.015%DMEM/Ham's F-12Friedman, S. and Roll, F.: Isolation and Culture of Hepatic Lipocytes,Kupffer Cells, and Sinusoidal Endothelial Cells by Density Gradient Centrifugation with Stractan,Anal Biochem 161, 207, 1987
Kupffer
Sinusoidal endothelial
大鼠wistar大鼠, 雄性, 3 个月EndothelialPronase: 0.25%HBSSNagelkenke, J., Barto, K., and VanBerkel, T.: In vivo and in vitro uptake and degradation of acetylated low density lipoprotein by rat liver endothelial, Kupffer, and parenchymal cells, J Biol Chem 258, 12221, 1983
Kupffer
Parenchymal
大鼠京都大鼠, 雄性, 100 - 200 克EndothelialCollagenase Type 2: 0.05%HBSSDiglio, C.A., Grammas, P., Filiberto Giacomelli, M.S., and Wiener, J.: Primary Culture of Rat Cerebral Microvascular Endothelial Cells, Lab Invest 46, 554, 1982
Cerebral
大鼠大鼠, 300 克, 和 猪, 30-40 公斤EndothelialTrypsin: 0.05%Medium 199 and 0.01M EDTAMerrilees, M.J., and Scott, L.: Interaction of aortic endothelial and smooth muscle cells in culture. Effect on glycosaminoglycan levels, Atherosclerosis 39, 147, 1981
Thoracic aorta
大鼠大鼠ForeskinCollagenase: 0.5%Dulbecco's MEM w/10% calf serumFolkman, J., Haudenschild, C. C., and Zetter, B. R.: Long-term Culture of Capillary Endothlial Cells, Proc Natl Acad Sci U S A 76, 5217, 1979
大鼠wistar大鼠, 成年的, 170 克EndothelialTrypsin: 0.5%BSSPhillips, P., Kumar, P., Kumar, S., and Waghe, M.: Isolation And Characterization of Endothelial Cells From Rat And Cow Brain White Matter, J Anat 129, 261, 1979

 

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