脑组织消化手册

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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，以便于制定一个*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
眼	心脏	肠	肾
肝	肺	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。

脑组织分离手册

物种	详细来源	细胞名称	消化用酶	培养基	参考文献
牛	牛	Microvascular endothelial	Neutral Protease: 0.005%	Medium 199	Kanda, T., Yoshino, H., Ariga, T., Yamawaki, M., and Yu, R.: Glycosphingolipid Antigens in Cultured Microvasular Bovine Brain Endothelial Cells: Sulfoglucuronosyl Paragloboside as a Target of Monoclonal IgM in Demyelinative Neuropathy, J Cell Biol 126 (1), 235, 1994
牛	牛	Brain endothelial cells	Collagenase Type 2: 0.35%	DMEM	Wolburg H, Neuhaus J, Kniesel U, Krauss B, Schmid EM, Ocalan M, Farrell C, Risau W: Modulation of tight junction structure in blood-brain barrier endothelial cells. Effects of tissue culture, second messengers and cocultured astrocytes, J Cell Sci 107, 1347, 1994
牛	牛	Endothelial	Neutral Protease: 0.125%	MEM	Miller, D., Audus, K., and Borchardt, R.: Application of Cultured Endothelial Cells of the Brain Microvasculature in the Study of the Blood-Brain Barrier, J Tiss Cul Meth 14, 217, 1992
牛	牛, 成年的	Cerebral artery	Collagenase: 0.2%	HBSS	Machi, 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
牛	牛, 成年的	Endothelial	Collagenase: 0.2%	HBSS
牛	牛	Capillary endothelial	Collagenase: 0.1%	DMEM	Estrada, C., Bready, J., Berliner, J., and Cancilla, P.: Choline Uptake by Cerebral Capillary Endothelial Cells in Culture, J Neurochem 54, 1467, 1990
牛	牛	Endothelial	Neutral Protease: 0.5%	MEM	Audus, K., and Borchardt, R.: Characterization of an In Vitro Blood-Brain Barrier Model System for Studying Drug Transport and Metabolism, Pharm Res 3 (2), 81, 1986
牛	牛	Endothelial	Collagenase Type 2: 0.2%	Dulbecco's PBS	Goetz, I.., Warren, J., Estrada, C., Roberts, E., and Krause, D.: Long-Term C*tion of Arterial and Capillary Endothelium From 成年的 Bovine Brain, In Vitro Cell Dev Biol 21, 172, 1985
牛	牛	Brain arteries	Collagenase Type 2: 0.2%	Dulbecco's PBS
牛	Calf	Oligodendroglia	Trypsin: 0.1%	(see reference)	Poduslo, S., Miller, K., and McKhann, G.: Metabolic Properties of Maintained Oligodendroglia Purified from Brain, J Biol Chem 253, 1592, 1978
牛	(also lamb)	Neural	Trypsin: 0.1%	(see reference)
豚鼠	豚鼠, 200-400 g	Neurons	Trypsin: 0.06-0.08%	PIPES saline	Kay, A.R., and Wong, R.K.S.: Isolation of Neurons Suitable for Patch-Clamping from 成年的 Mammalian Central Nervous Systems, J Neurosci Methods 16, 227, 1986
人类	人类	Tumor	Collagenase Type 4: 0.1%	see reference	Sauvageot, C., Weatherbee, J., Kesari, S., Winters, S., Barnes, J., Dellagatta, J., Ramakrishna, N., Stiles, C., Kung, A., Kieran, M. and Wen, P.: Efficacy of the HSP90 Inhibitor 17-AAG in Human Glioma Cell Lines and Tumorigenic Glioma Stem Cells.,Neuro Oncol Vol. 11,, , 109, 2009
			Hyaluronidase: 0.07%
			Deoxyribonuclease I: 0.04%
人类	人类	Microglia	Trypsin: 0.25%	DMEM/F12	Klegeris Andis, McGeer PatrickL: Chymotrypsin-like proteases contribute to human monocytic THP-1 cell as well as human microglial neurotoxicity, Glia 51, 56-64, 2005
人类	人类	Microglia	Deoxyribonuclease I: .005%	DMEM/F12
人类	人类	Microvessels	Collagenase Type 4: 0.1%	DMEM	Gerhart, 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	Neuronal	Deoxyribonuclease I: 10 礸/ml	Tris-HCl, 50 mM, CaCl2, 2 mM	Roher, A.E., Palmer, K.C., Chau, V., and Ball, M.J.: Isolation and Chemical Characterization of Alzheimer's Disease Paired Helical Filament Cytoskeletons:Differentiation from Amyloid Plaque Core Protein, J Cell Biol 107, 2703, 1988
人类	人类, 15-54 岁	Microvessels	Collagenase: 0.1%	Serum-free modified Lewis medium	Vinters, H.V., Reave, S., Coslo, P., Girvin, J.P., and Moore, S.A.: Isolation and Culture of Cells Derived From Human Cerebral Microvessels, Cell Tissue Res 249, 657, 1987
昆虫	果蝇	Neurons	Papain: 20 u/ml	Saline	Gu, H. and O'Dowd, D.: Whole Cell Recordings from Brain of 成年的 Drosophila., J Vis Exp 6, 248, 2007
昆虫	果蝇	Neurons	Papain: 4 u/ml	DMEM	Sicaeros, B. Campusano, J. and O'Dowd, D.: Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae., J Vis Exp 4, 200, 2007
猴	猴, 猕猴	Brain cells	Collagenase Type 2: 500 u/ml	HBSS	Marcondes MC, Burudi EM, Huitron-Resendiz S, Sanchez-Alavez M, Watry D, Zandonatti M, Henriksen SJ, Fox HS: Highly activated CD8(+) T cells in the brain correlate with early central nervous system dysfunction in simian immunodeficiency virus infection, J Immunol 167, 5429-38, 2001
猴	猴, 猕猴	Brain cells	Deoxyribonuclease I: 28 u/ml	HBSS
小鼠	小鼠	Chloroid plexus	Collagenase Type 4: 400 u/ml	PBS	Baruch, K., Rosenzweig, N., Kertser, A., Deczkowska, A., Sharif, A., Spinrad, A., Tsitsou-Kampeli, A., Sarel, A., Cahalon, L. and Schwartz, M.: Breaking Immune Tolerance by Targeting Foxp3(+) Regulatory T Cells Mitigates Alzheimer's Disease Pathology., Nat Commun 6, 7967, 2015
小鼠	小鼠	Brain-sequestered leukocytes	Collagenase Type 4: 0.05%	RPMI	Ryg-Cornejo, V., Ioannidis, L. and Hansen, D.: Isolation and Analysis of Brain-Sequestered Leukocytes from Plasmodium Berghei ANKA-Infected Mice., J Vis Exp71, e50112, 2013
小鼠	小鼠	Brain-sequestered leukocytes	Deoxyribonuclease I: 2 u/ml	RPMI
小鼠	小鼠	Cerebral pericytes	Collagenase Type 2: 0.1%	DMEM	Shah. G., Price, T., Banks, W., Morofuji, Y., Kovac, A., Ercal, N., Sorenson, C., Shin, E., Sheibani, N.: Pharmacological Inhibition of Mitochondrial Carbonic Anhydrases Protects Mouse Cerebral Pericytes from High Glucose-Induced Oxidative Stress and Apoptosis., J Pharmacol Exp Ther 344, 637-45, 2013
小鼠	小鼠	Cerebral pericytes	Deoxyribonuclease I: 30 u/ml	DMEM
小鼠	小鼠, P13-15	Glial	Papain: 20 u/ml	EBSS	Haseleu, J., Anlauf, E., Blaess, S., Endl, E. and Derouiche, A.: Studying Subcellular Detail in Fixed Astrocytes: Dissociation of Morphologically Intact Glial Cells (DIMIGs)., Front Cell Neurosci 7, 54, 2013
小鼠	小鼠, P13-15	Glial	Deoxyribonuclease I: 0.0005%	EBSS
小鼠	小鼠, 成年的	Neurons	PDS kit: with modifications	DMEM/F12	Saxena, A., Wagatsuma, A., Noro, Y., Kuji, T., Asaka-Oba, A., Watahiki, A., Gurnot, C., Fagiolini, M., Hensch, T. and Carninci, P.: Trehalose-Enhanced Isolation of Neuronal Sub-Types from 成年的 Mouse Brain., Biotechniques 52, 381, 2012
小鼠	小鼠, 1-2 天	Oligodendrocytes, dorsal root ganglia	Papain: 0.15%	DMEM	O'Meara, R., Ryan, S., Colognato, H. and Kothary, R.: Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-Cultures from Post-Natal Murine Tissues., J Vis Exp 54, 3324, 2011
小鼠	小鼠, 1-2 天	Oligodendrocytes, dorsal root ganglia	Deoxyribonuclease I: 0.006%	DMEM
小鼠	小鼠, 6-12周	Vascular smooth muscle cells	Papain: 0.05%	PBS	Chung, W., Farley, J., Swenson, A., Barnard, J., Hamilton, G., Chiposi, R. and Drummond, H.: Extracellular Acidosis Activates ASIC-like Channels in Freshly Isolated Cerebral Artery Smooth Muscle Cells., Am J Physiol Cell Physiol 298, C1198, 2010
			Collagenase Type 4: 0.15%
			Elastase: 0.05%
小鼠	小鼠, 4-6 天	Neurons	PDS kit: with modifications	HBSS	Lee, H., Greene, L., Mason, C. and Manzini, M.: Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons., J Vis Exp23, 990, 2009
小鼠	小鼠, 产后的	Astrocytes	Trypsin: 0.25%	HBSS	Sher, F., Rossler, R., Brouwer, N., Balasubramaniyan, V., Boddeke, E. and Copray, S.: Differentiation of Neural Stem Cells Into Oligodendrocytes: Involvement of the Polycomb Group Protein Ezh2., Stem Cells 26, 2875, 2008
小鼠	小鼠, 产后的	Astrocytes	Deoxyribonuclease I: 1,000 u/ml	HBSS
小鼠	小鼠, 初生的	Neurons	Papain: 20 u/ml	Neurobasal	Fasano, C., Thibault, D. and Trudeau, L.: Culture of Postnatal Mesencephalic Dopamine Neurons on an Astrocyte Monolayer,Current Protocols in Neuroscience Vol. 44,, , 3.21.1, 2008
小鼠	小鼠, 胚胎和出生后	Cortical neurons	Papain: 4-10 u/ml	Neurobasal	Hilgenberg, L. and Smith, M.: Preparation of Dissociated Mouse Cortical Neuron Cultures., J Vis Exp 10, 562, 2007
小鼠	小鼠	Neurosperes	PDS kit: per instructions	DMEM/F12	Klein C, Butt SJ, Machold RP, Johnson JE, and Fishell G.: Cerebellum- and forebrain-derived stem cells possess intrinsic regional character, Development132, 4497, 2005
小鼠	小鼠, 1 天	Neural progenitor	PDS kit: per instructions	see reference	Seaberg, R., Smukler, S. and Van der Kooy, D.: Intrinsic Differences Distinguish Transiently Neurogenic Progenitors from Neural Stem Cells in the Early Postnatal Brain., Dev Biol 278, 71, 2005
小鼠	小鼠	Granule cell precursors, pre-neoplastic and tumor cells	Papain: 10 u/ml	Neurobasal/B27	Oliver, T., Read, T., Kessler, J., Mehmeti, A., Wells, J., Huynh, T., Lin, S. and Wechsler-Reya, R.: Loss of Patched and Disruption of Granule Cell Development in a Pre-Neoplastic Stage of Medulloblastoma., Development 132, 2425, 2005
小鼠	小鼠	Granule cell precursors, pre-neoplastic and tumor cells	Deoxyribonuclease I: 250 u/ml	Neurobasal/B27
小鼠	小鼠	Cortical neurons	PDS kit: per instructions	Neurobasal	Hernandez, F., Perez, M., Lucas, J., Mata, A., Bhat, R. and Avila, J.: Glycogen Synthase Kinase-3 plays a Crucial Role in Tau Exon 10 Splicing and Intranuclear Distribution of SC35. Implications for Alzheimer's Disease., J Biol Chem 279, 3801, 2004
小鼠	小鼠, 3 天	Microglia	Papain: 90 u/ml	Eagle's MEM	Nishioku T, Hashimoto K, Yamashita K, Liou SY, Kagamiishi Y, Maegawa H, Katsube N, Peters C, von Figura K, Saftig P, Katunuma N, Yamamoto K, Nakanishi H: Involvement of cathepsin E in exogenous antigen processing in primary cultured murine microglia, J Biol Chem 277, 4816, 2002
小鼠	小鼠, 3 天	Microglia	Deoxyribonuclease I: 2000 u/ml	Eagle's MEM
小鼠	小鼠, SD, 8-12 周	Microglia	Trypsin: 0.125%	RPMI-1640	O'Donnell SL, Frederick TJ, Krady JK, Vannucci SJ, Wood TL: IGF-I and microglia/macrophage proliferation in the ischemic mouse brain, Glia 39, 85, 2002
			Collagenase Type 2: 0.01%
			Deoxyribonuclease I: .005%
小鼠	小鼠, 2-5天	Postnal substantia nigra	PDS kit: per instructions	see reference	Smeyne Michelle, Smeyne RichardJ: Method for culturing postnatal substantia nigra as an in vitro model of experimental Parkinson's disease, Brain Res Brain Res Protoc 9, 105-11, 2002
小鼠	小鼠, 胚胎	Cortical progenitors	PDS kit: per instructions	Serum free medium	Estivill-Torrus, G., Pearson, H., Van Heyningen, V., Price, D. and Rashbass, P.:Pax6 is Required to Regulate the Cell Cycle and the Rate of Progression from Symmetrical to Asymmetrical Division in Mammalian Cortical Progenitors.,Development 129, 455, 2002
小鼠	小鼠, 1 天	Neurons and glia	PDS kit: per instructions	Neurobasal	Martin-Aparicio, E., Yamamoto, A., Hernandez, F., Hen, R., Avila, J. and Lucas, J.: Proteasomal-Dependent Aggregate Reversal and Absence of Cell Death in a Conditional Mouse Model of Huntington's Disease., J Neurosci 21, 8772, 2001
小鼠	小鼠	Cerebellar granule cell precursors	Papain: 0.435%	EBSS	Miyazawa K, Himi T, Garcia V, Yamagishi H, Sato S, and Ishizaki Y.: A role for p27/Kip1 in the control of cerebellar granule cell precursor proliferation, J Neurosci 20, 5756, 2000
小鼠	小鼠	Cerebellar granule cell precursors	Deoxyribonuclease I: 0.05%	EBSS
小鼠	小鼠	Astrocytes	Trypsin: 0.25%	DMEM	Lim, D, and Alvarez-Buylla, A.: Interaction Between Astrocytes and 成年的 Subventricular Zone Precursors Stimulates Neurogenesis, Proc Natl Acad Sci U S A 96, 7526, 1999
小鼠	小鼠, 初生的	Hippocampal cells	Papain: 10 u/ml	DMKM	Jun K, Choi G, Yang SG, Choi KY, Kim H, Chan GC, Storm DR, Albert C, Mayr GW, Lee CJ, Shin HS: Enhanced hippocampal CA1 LTP but normal spatial learning in inositol 1,4,5-trisphosphate 3-kinase(A)-deficient mice, Learn Mem 5, 317-30, 1998
小鼠	小鼠, SWR or CF1, 1-3 个月	Papillae, taste receptor	Pronase E: 0.15%	Carbonate-Phosphate buffer	Spielman, A., Mody, I., Brand, J., Whitney, G., MacDonald, J., and Salter, M.: A Method for Isolating and Patch-Clamping Single Mammalian Taste Receptor Cells,Brain Res 503, 326, 1989
小鼠	小鼠, SWR or CF1, 1-3 个月	Papillae, taste receptor	Pronase E: 0.15%	(see reference)
小鼠	小鼠, 0-30 天	Neural	Trypsin NF 1:250: 50 0.25%	BSS	Shrier, B., Wilson, S., and Nirenberg, M.: Cultured Cell Systems and Methods for Neurobiology, Vol. 32,, , 765, 1974
绵羊	羔羊	Oligodendroglia	Trypsin: 0.1%	(see reference)	Poduslo, S., Miller, K., and McKhann, G.: Metabolic Properties of Maintained Oligodendroglia Purified from Brain, J Biol Chem 253, 1592, 1978
绵羊	(还有牛犊)	Neural	Trypsin: 0.1%	(see reference)
猪	小型猪, 尤卡坦半岛 (尤卡坦半岛野猪), 4-6 个月	Microvascular	Collagenase: 0.1%	HBSS	Robinson, D.H., Kang,Y., Deschner, S.H., and Nielsen, T.B.: Morphologic Plasticity and Periodicity : Porcine Cerebral Microvascular Cells in Culture, In Vitro Cell Dev Biol 26, 169, 1990
大鼠	大鼠, SD, 19-21 天	Suprachiasmatic nucleus neurons	Papain: 100 u/ml	MEM	Cao, R., Li, A., Cho, H., Lee, B. and Obrietan, K.: Mammalian Target of Rapamycin Signaling Modulates Photic Entrainment of the Suprachiasmatic Circadian Clock., J Neurosci 30, 6302, 2010
大鼠	大鼠, 食鱼动物, 7-21 个月	Hippocampal neurons	Papain: 0.2%	Hibernate A	Chen, N., Newcomb, J., Garbuzova-Davis, S., Davis Sanberg, C., Sanberg, P. and Willing, A.: Human Umbilical Cord Blood Cells Have Trophic Effects on Young and Aging Hippocampal Neurons in Vitro., Aging Dis 1, 173, 2010
大鼠	大鼠, SD, 7 天	Cerebellar granule neurons	PDS kit: per instructions	PBS	Tanaka, S., Shaikh, I., Chiocca, E. and Saeki, Y.: The Gs-Linked Receptor GPR3 Inhibits the Proliferation of Cerebellar Granule Cells During Postnatal Development., PLoS ONE 4, e5922, 2009
大鼠	大鼠, neonatal	Astrocytes	Papain: 20 u/ml	EBSS	Shigetomi, E. and Khakh, B.: Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes., J Vis Exp 26, 1142, 2009
大鼠	大鼠,初生的	Hippocampal neurons	Papain: 10 u/ml	EBSS	Richler Esther, Chaumont Severine, Shigetomi Eiji, Sagasti Alvaro, Khakh Baljit S: Tracking transmitter-gated P2X cation channel activation in vitro and in vivo,Nat Methods 5, 87-93, 2008
大鼠	大鼠, SD	Hippocampal	Trypsin: 0.1%	Neurobasal	Akanda, N., Tofighi, R., Brask, J., Tamm, C., Elinder, F. and Ceccali, S.:Voltage-Dependent Anion Channels (VDAC) in the Plasma Membrane Play a Critical Role in Apoptosis in Differentiated Hippocampal Neurons but not in Neural Stem Cells., Cell Cycle 7, 3225, 2008
大鼠	大鼠, neonatal	Dura mater	Collagenase Type 1: 0.075%	DMEM	Peptan, I., Hong, L. and Evans, C.: Multiple Differentiation Potentials of Neonatal Dura Mater-Derived Cells., Neurosurgery 60, 346, 2007
大鼠	大鼠, E18	Hippocampal neurons	Papain: 0.2%	Hibernate	Jekabsons MB, Nicholls DG: Bioenergetic analysis of cerebellar granule neurons undergoing apoptosis by potassium/serum deprivation, Cell Death Differ 13, 1595-610, 2006
大鼠	大鼠, 威斯塔, 1-3 day	Hippocampal neurons	Trypsin: 0.05%	DMEM	Velasco Myrian, Garci璦 Esperanza, Onetti Carlos G: Glucose deprivation activates diversity of potassium channels in cultured rat hippocampal neurons,Cell Mol Neurobiol 26, 307-19, 2006
大鼠	大鼠, E19	Hippocampal neuron	Papain: 10 u/ml	MEM	Khakh Baljit S, Fisher James A, Nashmi Raad, Bowser David N, Lester Henry A:An angstrom scale interaction between plasma membrane ATP-gated P2X2 and alpha4beta2 nicotinic channels measured with fluorescence resonance energy transfer and total internal reflection fluorescence microscopy, J Neurosci 25, 6911-20, 2005
大鼠	大鼠, 威斯塔, 14天	Visual cortical	PDS kit: per instructions	EBSS	Mizoguchi Y, Kanematsu T, Hirata M, Nabekura J: A rapid increase in the total number of cell surface functional GABAA receptors induced by brain-derived neurotrophic factor in rat visual cortex, J Biol Chem 278, 44097, 2003
大鼠	大鼠, 威斯塔, 雄性	Cerebral endothelial	Collagenase Type 3: 0.2%	MEM	Floris S, Van den Born J, van der Pol SM, Dijkstra CD, De Vries HE: Heparan sulfate proteoglycans modulate monocyte migration across cerebral endothelium, J Neuropathol Exp Neurol 62, 780, 2003
大鼠	大鼠, SD, E18	Cortical cells	Papain: 20 u/ml	EBSS	Behar, T, Smith, S, Kennedy, R, Mckenzie, J, Maric, I and Barker, J: GABA(B) receptors mediate motility signals for migrating embryonic cortical cells, Cereb Cortex 11, 744-53, 2001
大鼠	大鼠, SD, E18	Cortical cells	Deoxyribonuclease I: .005%	EBSS
大鼠	大鼠, 威斯塔, 250-300 g	Cerebral artery smooth muscle cells	Papain: 1.5 mg/ml	Physiological Salt Solution	Brzezinska AK, Gebremedhin D, Chilian WM, Kalyanaraman B, Elliott SJ.:Peroxynitrite reversibly inhibits Ca(2+)-activated K(+) channels in rat cerebral artery smooth muscle cells, Am J Physiol Heart Circ Physiol 278, H1883, 2000
大鼠	大鼠, 威斯塔, 250-300 g	Cerebral artery smooth muscle cells	Collagenase Type 4: 1.5 mg/ml	Physiological Salt Solution
大鼠	大鼠, SD, E16	Cortical neurons	PDS kit: with modifications	Neurobasal Medium	Varney MA, Cosford ND, Jachec C, Rao SP, Sacaan A, Lin FF, Bleicher L, Santori EM, Flor PJ, Allgeier H, Gasparini F, Kuhn R, Hess SD, Velicelebi G, Johnson EC:SIB-1757 and SIB-1893: selective, noncompetitive antagonists of metabotropic glutamate receptor type 5, J Pharmacol Exp Ther 290, 170, 1999
大鼠	大鼠, 威斯塔, 初生的	Hippocampal cells	Papain: 1 mg/ml	DMEM	Boehm S, Betz H: Somatostatin inhibits excitatory transmission at rat hippocampal synapses via presynaptic receptors, J Neurosci 17, 4066, 1997
大鼠	大鼠, SD, 食鱼动物	Hippocampal neurons	Papain: 0.2%	HibernateA/B27	Brewer, G.J.: Isolation and culture of 成年的 rat hippocampal neurons of any age, J Neurosci Methods 71, 143, 1997
大鼠	大鼠, E18	Hippocampal and cortical neurons	PDS kit: per instructions	Neurobasal media	Naeve GS, Ramakrishnan M, Kramer R, Hevroni D, Citri Y, Theill LE: Neuritin: a gene induced by neural activity and neurotrophins that promotes neuritogenesis,Proc Natl Acad Sci U S A 94, 2648-53, 1997
大鼠	大鼠, SD, 初生	Glial	Trypsin: 0.0625%	MEM, sterile	Pixley, S.K.: The Olfactory Nerve Contains Two Populations of Glia, In Vitro 5, 269, 1992
大鼠	大鼠, 初生	Astrocytes	Trypsin: 0.25%	DMEM, HBSS	Holzwarth, J., Glaum, S., and Miller, R.: Activation of Endothelin Receptors by Sarafotoxin Regulates Ca2+ Homeostasis in Cerebellar Astrocytes, Glia 5, 239, 1992
大鼠	大鼠, 60-72 小时大的	Fibroblasts	Trypsin: 0.2%	HEPES buffered DMEM	Acheson, A., Barker, P., Alderson, R., Miller, F., and Murphy, R.: Detection of Brain-Derived Neurotrophic Factor-like Activity in Fibroblasts and Schwann Cells: Inhibition by Antibodies to NGF, Neuron 7, 265, 1991
大鼠	大鼠, SD, 成年的	Endothelial	Collagenase Type 2: 0.5%	Medium 199	Doron, D., Jacobowitz, D., Heldman, E., Feurerstein, G., Pollard, H., and Hallenbeck, J.: Extracellular Matrix Permits the Expression of Von Willebrand's Factor, Uptake of Di-I-Acetylated Low Density Lipoprotein and Secretion of Prostacyclin in Cultures of Endothelial Cells from Rat Brain Microvessels, In Vitro Cell Dev Biol 27A, 689, 1991
大鼠	大鼠, SD, 5天	Hippocampal neurons	Trypsin: 0.2%	HBSS	Mattson, M., and Kater, S.: Development and Selective Neurodegeneration in Cell Cultures From Different Hippocampal Regions, Brain Res 490, 110, 1989
大鼠	大鼠, SD, 5天	Hippocampal neurons	Deoxyribonuclease I: 10 ug/ml	HBSS
大鼠	大鼠, SD, 雌性	Fetal rat brain	Collagenase Type 4: 0.1%	DMEM	Matsuda, M.: Serum Proteins Enhance Aggregate Formation of Dissociated Fetal Rat Brain Cells in an Aggregating Culture, In Vitro Cell Dev Biol 24 (10), 1031, 1988
大鼠	大鼠, 白化病, 成年的和初生的	Cerebral cortices	Trypsin: 0.25%	PBS	Giulian, D. and Baker, T.J.: Characterization of Ameboid Microglia Isolated From Developing Mammalian Brain, J Neurosci 6, 2163, 1986
大鼠	大鼠, 威斯塔, 初生	Germinal matrix	Trypsin: 0.25%	HBSS	Goldman, J.E., Geier, S.S., and Hirano, M.: Differentiation of Astrocytes and Oligodendrocytes From Germinal Matrix Cells in Primary Culture, J Neurosci 6, 52, 1986
大鼠	大鼠,胎儿, 18-20天	Hippocampal neurons	Trypsin: 0.25%	HBSS, CMF	Bartlett, W. and Banker, G.: An Electron Microscopic Study of the Development of Axons and Dendrites by Hippocampal Neurons in Culture. I. Cells Which Develop Without Intercellular Contacts, J Neurosci 4, 1944, 1984
大鼠	大鼠, 胎儿, 18 天	Hippocampi	Trypsin: 0.1%	HBSS	Rothman, S.: Synaptic Release of Excitatory Amino Acid Neurotransmitter Mediates Anoxic Neuronal Death, J Neurosci 4 (7), 1884, 1984
大鼠	大鼠, SD, 怀孕19-20天的	Neural	Trypsin: 0.25%	DMEM	Ahmed, Z., Walker, P., and Fellows, R.: Properties of Neurons from Dissociated Fetal Rat Brain in Serum-Free Culture, J Neurosci 3 (12), 2448, 1983
大鼠	大鼠, SD, 胎儿	Cerebral cortex	Deoxyribonuclease I: 0.001%	HEPES	Peterfreund, R. and Vale, W.: High Molecular Weight Somatostatin Secretion by Cultured Rat Brain Cell, Brain Res 239, 463, 1982
大鼠	大鼠, SD, 胎儿	Hypothalmus	Deoxyribonuclease I: 0.001%	HEPES
大鼠	Wistar京都大鼠, 雄性, 100 - 200 克	Endothelial	Collagenase Type 2: 0.05%	HBSS	Diglio, C.A., Grammas, P., Filiberto Giacomelli, M.S., and Wiener, J.: Primary Culture of Rat Cerebral Microvascular Endothelial Cells, Lab Invest 46, 554, 1982
大鼠	Wistar京都大鼠, 雄性, 100 - 200 克	Cerebral	Collagenase Type 2: 0.05%	HBSS
大鼠	大鼠, 胎儿	Cortical	Trypsin: 0.25%	Puck's D1 (see reference)	Swaiman, K., Neale, E., Fitzgerald, S., and Nelson, P.: A Method for Large-scale Production of Mouse Brain Cortical Cultures, Brain Res 255, 361, 1982
大鼠	大鼠, 威斯塔,怀孕的	Glial	Trypsin: 0.05%	Eagle's MEM/DMEM	Abney, E., Bartlett, P., and Raff, M.: Astrocytes, Ependymal Cells, and Oligodendrocytes Develop on Schedule in Dissociated Cell Cultures of Embryonic Rat Brain, Dev Biol 83, 301, 1981
大鼠	大鼠, SD, 一个月大的	Capillary endothelium	Neutral Protease: 0.1%	Medium 199	Bowman, P., Betz, A., Ar, D., Wolinsky, J., Penney, J., Shivers, R., and Goldstein, G.: Primary Culture of Capillary Endothelium From Rat Brain, In Vitro 17 (4), 353, 1981
大鼠	大鼠, SD, 一个月大的	Pericytes	Neutral Protease: 0.1%	Medium 199
大鼠	大鼠, 威斯塔, 雄性, 300-500 克	Microvessels	Collagenase Type 2: 0.75%	Ringers-HEPES buffer	Williams, S., Gillis, J., Matthews, M., Wagner, R., and Bitensky, M.: Isolation and Characterization of Brain Endothelial Cells: Morphology and Enzyme Activity, J Neurochem 35 (2), 374, 1980
大鼠	大鼠, 威斯塔, 雄性, 300-500 克	Endothelial	Collagenase Type 2: 0.75%	Ringers-HEPES buffer
大鼠	大鼠, 威斯塔, 成年的, 170克	Endothelial	Trypsin: 0.5%	BSS	Phillips, 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
大鼠	大鼠, 豪斯曼, 18 天	Hippocampal neurons	Trypsin: 0.1%	HBSS	Banker, G., and Cowan, M.: Rat Hippocampal Neurons in Dispersed Cell Culture,Brain Res 126, 397, 1977
大鼠	大鼠, SD, 2 天	Pineal	Trypsin: 0.25%	DMEM, MEM	Rowe, V., Neale, E., Avins, L., Guroff, G., and Schrier, B.: Pineal gland cells in culture. Morphology, Biochemistry, Differentiation, and co-culture with sympathetic neurons, Exp Cell Res 104, 345, 1977
贝类动物	旋节螺trivolvis	Neurons	Trypsin: 0.2%	Saline, Sterile	Hadley, R.D., Bodnar, D.A., and Kater, S.B.: Formation of Electrical Synapses Between Isolated, Cultured Helisoma Neurons Requires Mutual Neurite Elongation,J Neurosci 5, 3145, 1985
贝类动物	旋节螺trivolvis	Buccal ganglia	Trypsin: 0.2%	Saline, Sterile

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