腎上腺組織分離手冊

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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(yǎng)和細胞生物學(xué)研究的原代細胞的分離和收集。盡管這些消化酶已經(jīng)普遍應(yīng)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(dǎo)致的結(jié)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(jīng)驗，而不是在*理解消化原理的基礎(chǔ)上選擇的消化方案，以及如何根據(jù)具體實驗?zāi)康男拚桨敢垣@得更好的實驗結(jié)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(jié)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(yǎng)液

6、所用的酶

7、消化酶中的雜質(zhì)

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(jīng)驗決定以獲取zui適實驗結(jié)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(shù)據(jù)搞得一頭霧水。這些巨大差異來源于組織胞外基質(zhì)的復(fù)雜而動態(tài)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(zhì)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(fā)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(jié)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(guān)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(jié)腸組織	內(nèi)皮組織	上皮組織
眼	心臟	腸	腎
肝	肺	淋巴結(jié)	乳腺
其它組織	其他組織	肌組織	神經(jīng)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。

腎上腺組織分離手冊

物種	詳細來源	細胞名稱	消化用酶	培養(yǎng)基	參考文獻
牛	牛	Chromaffin	Collagenase Type 1: 0.1-0.2%	DMEM/F12	Dominguez, N., Rodri璯uez, M., Machado, J. and Borges, R.: Preparation and Culture of Adrenal Chromaffin Cells., Methods Mol Biol 846, 223, 2012
			Deoxyribonuclease I: 0.003-0.015%
			Hyaluronidase: 0.015%
牛	牛, 6 個月	Chromaffin	Collagenase Type 1: 0.125%	Locke's solution	Moustafa T, Girod S, Tortosa F, Li R, Sol JC, Rodriguez F, Bastide R, Lazorthes Y, Sallerin B: Viability and functionality of b綿羊 chromaffin cells encapsulated into alginate-PLL microcapsules with a liquefied inner core, Cell Transplant 15, 121-33, 2006
牛	牛	Chromaffin cells	Collagenase: 0.1%	DMEM	Hahm, S., Chen, Y., Vinson, C. and Eiden, L.: A Calcium-Initiated Signaling Pathway Propagated Through Calcineurin and cAMP Response Element-Binding Protein Activates Proenkephalin Gene Transcription after Depolarization., Mol Pharmacol 64, 1503, 2003
牛	牛	Chromaffin cells	Deoxyribonuclease I: 30 u/ml	DMEM
牛	牛	Chromaffin	Collagenase: 0.2%	Locke's solution	Ortega, J., Sagen, J., and Pappas, G.: Short-term Immunosuppression Enhances Long-term Survival of B綿羊 Chromaffin Cell Xenografts in Rat CNS,Cell Transplant 1, 33, 1992
牛	牛	Chromaffin	Deoxyribonuclease I: 30 u/mg	HEPES	Zhu, J., Li, W., Toews, M., and Hexum, T.: Neuropeptide Y Inhibits Forskolin-Stimulated Adenylate Cyclase in B綿羊 Adrenal Chromaffin Cells via a Pertussis Toxin-Sensitive Process, J Pharmacol Exp Ther 263 (3), 1479, 1992
牛	牛	Heart	Trypsin: 0.06%	25mM HEPES buffered Locke's solution, CMF	Trifaro, J., Tang, R., and Novas, M.: Monolayer Co-Culture of Rat Heart Cells and B綿羊 Adrenal Chromaffin Paraneurons, In Vitro Cell Dev Biol 26, 335, 1990
	(also 大鼠)	Adrenal chromaffin
		Paraneurons
牛	牛	Chromaffin	Collagenase Type 1: 0.25 %	DMEM	Dahmer, M., Hart, P., and Perlman, R.: Studies on the Effect of Insulin-Like Growth Factor-I on Catecholamine Secretion from Chromaffin Cells, J Neurochem 54 (3), 931, 1990
牛	牛	Chromaffin	Collagenase: 0.2%	Locke's solution	Higgins, L., and Berg, D.: Immunological Identification of a Nicotinic Acetylcholine Receptor on B綿羊 Chromaffin Cells, J Neurosci 7 (6), 1792, 1987
牛	牛	Chromaffin	Deoxyribonuclease I: 0.015%	Locke's solution
牛	牛	Chromaffin	Collagenase: 0.05%	Locke's solution, CMF	Aunis, D., Rotllan, P., and Miras-Portugal, M.: Incorporation of Adenosine into Nucleotides of Chromaffin Cells in Culture, Neurochem Int 7, 89, 1985
牛	牛	Chromaffin	Collagenase: 0.15%	Kreb's, CMF	Almazan, G., Aunis, D., Garcia, A., Montiel, C., Nicolas, G., and Sanchez-Garcia, P.: Effects Of CLS on the Release of Noradrenaline From Chromaffin Cells, Br J Biomed Sci 81, 599, 1984
牛	牛	Chromaffin	Collagenase: 0.1%	(see reference)	Pollard, H., Pazoles, C., Creutz, C., Scott, J., Zinder, O., and Hotchkiss, A.:An Osmotic Mechanism For Exocytosis From Dissociated Chromaffin Cells, J Biol Chem 259, 1114, 1984
牛	牛	Chromaffin	Collagenase Type 1: 0.05%	CF Kreb's	Cena, V., Garcia, A., Montiel, C., and Sanchez-Garcia, P.: Uptake of [3H]-nicotine and [3H]-noradrenaline by Cultured Chromaffin Cells, Br J Pharmacol 81, 119, 1984
牛	牛	Chromaffin	Collagenase Type 1: 0.025%	HBSS, modified	Waymire, J., Bennett, W., Boehme, R., Hankins, L., Gilmer-Waymire, K., and Haycock, J.: B綿羊 Adrenal Chromaffin Cells: High-Yield Purification and Viability in Suspension Culture, J Neurosci Methods 7, 329, 1983
牛	牛	Medulla	Hyaluronidase: 0.2%	Saline w/BSA 0.5%	Knight, D. and Baker, P.: Stimulus-Secretion Coupling in Isolated B綿羊 Adrenal Medullary Cells, Q J Exp Physiol 68, 123, 1983
牛	牛	Medulla	Collagenase: 0.2%	Krebs-Ringer bicarbonate buffer, CMF	Greenberg, A. and Zinder, O.: alpha- and beta-Receptor Control of Catecholamine Secretion from Isolated Adrenal Medulla Cells, Cell Tissue Res226, 655, 1982
牛	牛	Chromaffin	Deoxyribonuclease I: 15 礸/ml	Medium A	Wilson, S.P., and Viveros, O.H.: Primary Culture of Adrenal Medullary Chromaffin Cells in a Chemically Defined Medium, Exp Cell Res 133, 159, 1981
牛	牛	Chromaffin	Deoxyribonuclease I: 15 礸/ml	(see reference)
牛	牛	Medulla	Protease: 0.2%	Saline	Baker, P., and Knight., D: Calcium Control of Exocytosis and Endocytosis in B綿羊 Adrenal Medullary Cells, Phil Trans R Soc Lond 296, 83, 1981
牛	牛	Chromaffin	Collagenase: 0.05%	Locke's solution, CMF	Trifaro, J.M., and Lee, R.W.: Morphological Characteristics and Stimulus-Secretion Coupling in B綿羊 Adrenal Chromaffin Cell Cultures, Neuroscience 5, 1533, 1980
牛	牛, 成年的	Chromaffin	Collagenase: 0.5%	HBSS	Unsicker, K., Rieffert, B., and Ziegler, W.: Effects of Cell Culture Conditions, Nerve Growth Factor, Dexamethasone, and Cyclic AMP on Adrenal Chromaffin Cells In Vitro, Adv Biochem Psychopharmacol 255, 51, 1980
牛	(還有大鼠, 加拿大-威斯塔, 年幼的; 豚鼠, 初生)	Chromaffin	Collagenase: 0.5%	HBSS
牛	牛	Chromaffin	Collagenase: 0.25%	F-12 medium	Kumakura, K., Karoum, F., Guidotti, A., and Costa, E.: Modulation of Nicotinic Receptors by Opiate Receptor Agonists in Cultured Adrenal Chromaffin Cells, Nature 283, 489, 1980
牛	牛	Medulla	Collagenase: 0.05%	Locke's solution, CF	Kilpatrick, D., Ledbetter, F., Carson, K., Kirshner, A., Slepetis, R., and Kirshner, N.: Stability of B綿羊 Adrenal Medulla Cells in Culture, J Neurochem 35 (3), 679, 1980
牛	牛, 成年的	Medulla	Collagenase Type 1: 0.5%	HBSS	Unsicker, K., and Griesser, G.: Establishment, Characterization and Fibre Outgrowth of Isolated B綿羊 Adrenal Medullary Cells in Long-Term Cultures,Neuroscience 5, 1445, 1980
牛	牛	Chromaffin	Collagenase: 0.05%	DMEM	Aunis, D., Guerold, B., Bader, M-F., and Cieselski-Treska, J.:Immunocytochemical and Biochemical Demonstration of Contractile Proteins in Chromaffin Cells, Neuroscience 5, 2261, 1980
牛	牛	Medullary	Collagenase Type 2: 0.2%	HEPES	Hersey, R., and DiStefano, V.: Control of Phenylethanolamine N-Methyltransferase by Glucocorticoids in Cultured B綿羊 Adrenal Medullary Cells, J Pharmacol Exp Ther 209 (1), 147, 1979
牛	牛	Foreskin	Collagenase: 0.5%	Dulbecco's MEM w/10% calf serum	Folkman, 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
牛	牛	Medulla	Collagenase Type 1: 0.05%	Kreb's, CF	Fenwick, E., Fajdiga, P., Howe, N., and Livett, B.: Functional and Morphological Characterization of Isolated B綿羊 Adrenal Medullary Cell, J Cell Biol 76, 12, 1978
牛	牛	Chromaffin	Hyaluronidase: 0.2%	HEPES, CF	Brooks, J.C.: The Isolated B綿羊 Adrenomedullary Chromaffin Cell: A Model of Neuronal Excitation-Secretion, Endocrinology 101, 1369, 1977
牛	牛, 雄性	Chromaffin	Collagenase Type 1: 0.2%	Kreb's Ringer bicarbonate buffer, CF	Schneider, A., Herz, R., and Rosenheck, K.: Stimulus-Secretion Coupling in Chromaffin Isolated From Adrenal Medulla, Proc Natl Acad Sci U S A 74, 5036, 1977
豚鼠	豚鼠, 500-700克	Chromaffin	Collagenase:	BSS	Role, L.W., Leeman, S.E., and Perlman, R.L.: Somatostain and Substance P Inhibit Catecholamine Secretion from Isolated Cells of 豚鼠 Adrenal Medulla, Neurochem Int 6, 1813, 1981
豚鼠	豚鼠, 500-700克	Medulla	Collagenase:	(see reference)
豚鼠	豚鼠	Chromaffin	Collagenase: 0.5%	HBSS	Unsicker, K., Rieffert, B., and Ziegler, W.: Effects of Cell Culture Conditions, Nerve Growth Factor, Dexamethasone, and Cyclic AMP on Adrenal Chromaffin Cells In Vitro, Adv Biochem Psychopharmacol 255, 51, 1980
豚鼠	(還有大鼠,漢諾威-威斯塔, 年幼的; 初生; 牛)	Chromaffin	Collagenase: 0.5%	HBSS
豚鼠	豚鼠	Adrenal	Collagenase: 0.05%-0.20%	Kreb's-Ringer bicarb glucose buffer, CF	Hochman, J., and Perlman, R.L.: Catecholamine Secretion by Isolated Adrenal Cells, Biochim Biophys Acta 421, 168, 1976
豚鼠	豚鼠	Chromaffin	Collagenase: 0.05%-0.20%	Kreb's-Ringer bicarb glucose buffer, CF
倉鼠	倉鼠 (金倉鼠) 100-150 克	Adrenal	Hyaluronidase: 0.20%	Kreb's Ringer bicarbonate buffer	Liang, B.T., and Perlman, R.L.: Catecholamine Secretion by 倉鼠 Adrenal Cells, J Neurochem 32, 927, 1979
倉鼠	倉鼠 (金倉鼠) 100-150 克	Chromaffin	Hyaluronidase: 0.20%	Kreb's Ringer bicarbonate buffer
人類	人類	Adrenal medulla progenitor	Collagenase: 0.2%	DMEM/F-12	Santana, M., Chung, K., Vukicevic, V., Rosmaninho-Salgado, J., Kanczkowski, W., Cortez, V., Hackmann, K., Bastos, C., Mota, A., Schrock, E., Bornstein, S., Cavadas, C. and Ehrhart-Bornstein, M.: Isolation, Characterization, and Differentiation of Progenitor Cells from 人類 Adult Adrenal Medulla., Stem Cells Transl Med 1, 783, 2012
人類	人類	Chromaffin cells	Collagenase: 0.2%	Locke's solution	Jeon, Y., Baek, W., Chung, S., Shin, N., Kim, H., and Lee, S.: Cultured 人類 Chromaffin Cells Grafted in Spinal Subarachnoid Space Relieves Allodynia in a Pain Rat Model., Korean J Anesthesiol 60, 357, 2011
人類	人類	Adrenocortical	Collagenase Type 1: 0.2%	Krebs Ringer	Caroccia, B., Fassina, A., Seccia, T., Recarti, C., Petrelli, L., Belloni, A., Pelizzo, M. and Rossi, G.: Isolation of 人類 Adrenocortical Aldosterone-Producing Cells by a Novel Immunomagnetic Beads Method., Endocrinology 151, 1375, 2010
人類	人類	Adrenocortical	Deoxyribonuclease I: 0.01%	Krebs Ringer
人類	人類, 成年的	Chromaffin	Trypsin: 0.25%	Eagle's MEM	Tischler, A., DeLellis, R., Bailes, B., Nunnemacher, G.,Carabba, V., and Wolfe, H.: Nerve Growth Factor-Induced Neurite Outgrowth from Normal 人類 Chromaffin Cells, Lab Invest 43, 399, 1980
人類	人類, 成年的和小孩	Foreskin	Collagenase: 0.5%	Dulbecco's MEM w/10% calf serum	Folkman, 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
小鼠	小鼠	Chromaffin	Collagenase Type 1: 0.1-0.2%	DMEM/F12	Dominguez, N., Rodri璯uez, M., Machado, J. and Borges, R.: Preparation and Culture of Adrenal Chromaffin Cells., Methods Mol Biol 846, 223, 2012
			Deoxyribonuclease I: 0.003-0.015%
			Hyaluronidase: 0.015%
小鼠	小鼠, 8-10 周	Chromaffin	Papain: 40 u/ml	DMEM	Kolski-Andreaco, A., Cai, H., Currle, D., Chandy, K. and Chow, R.: 小鼠 Adrenal Chromaffin Cell Isolation., J Vis Exp 2, 129, 2007
小鼠	小鼠, 胚胎	Chromafin cells	Papain: 20-25 u/ml	DMEM	Tian Jin-Hua, Wu Zheng-Xing, Unzicker Michael, Lu Li, Cai Qian, Li Cuiling, Schirra Claudia, Matti Ulf, Stevens David, Deng Chuxia, Rettig Jens, Sheng Zu-Hang: The role of Snapin in neurosecretion: snapin knock-out mice exhibit impaired calcium-dependent exocytosis of large dense-core vesicles in chromaffin cells, J Neurosci 25, 10546-55, 2005
綿羊	綿羊, 成年的和胚胎	Adrenocortical	Collagenase Type 1: 0.4%	DMEM/Ham's F12	Valego, N. and Rose, J.: A Specific CRH Antagonist Attenuates ACTH-Stimulated Cortisol Secretion in 綿羊 Adrenocortical Cells., Reprod Sci 17, 477, 2010
綿羊	綿羊, 胚胎	Adrenocortical	Collagenase Type 1: 0.4%	DMEM/Ham's F12	Valego, N., Su, Y., Carey, L., Young, S., Tatter, S., Wang, J. and Rose, J.:Hypothalamic-Pituitary Disconnection in Fetal Sheep Blocks the Peripartum Increases in Adrenal Responsiveness and Adrenal ACTH Receptor Expression.,Am J Physiol Regul Integr Comp Physiol 289, R410, 2005
綿羊	綿羊, 成年	Chromaffin cells	Collagenase Type 2: 0.2%	Locke's solution	Keating, D., Rychkov, G., Adams, M., Holgert, H., McMillen, I.C. and Roberts, M.: Opioid Receptor Stimulation Suppresses the Adrenal Medulla Hypoxic Response in Sheep by Actions on Ca(2+) and K(+) Channels., J Physiol555, 489, 2004
綿羊	綿羊, 成年	Chromaffin cells	Deoxyribonuclease I: 100 u/ml	Locke's solution
綿羊	綿羊, 3 年	Anterior pituitary	Trypsin: 2.5%	DMEM	Canny B J, O'Farrell K A, Clarke I J, Tilbrook A J: The influence of sex and gonadectomy on the hypothalamo-pituitary-adrenal axis of the sheep, J Endocrinol 162, 215-25, 1999
綿羊	綿羊, 3 年	Anterior pituitary	Deoxyribonuclease I: 0.004%	DMEM
大鼠	大鼠	Chromaffin	Collagenase Type 1: 0.1-0.2%	DMEM/F12	Dominguez, N., Rodri璯uez, M., Machado, J. and Borges, R.: Preparation and Culture of Adrenal Chromaffin Cells., Methods Mol Biol 846, 223, 2012
			Deoxyribonuclease I: 0.003-0.015%
			Hyaluronidase: 0.015%
大鼠	大鼠	Chromaffin	Collagenase Type 1: 0.26%	HBSS	Gilabert, J, Montalvo, G, and Artalejo A.: Rat Chromaffin Cells Primary Cultures: Standardization and Quality Assessment for Single-Cell Assays,Nat Protoc , 294, 2006
			Deoxyribonuclease I: 0.015%
			Hyaluronidase: 0.015%
大鼠	大鼠, SD	Chromaffin cells	Collagenase Type 1: 0.26%	HBSS	Gilabert, J: Necessary Conditions to Maintain Rat Adrenal Chromaffin Cells in Primary Culture,Cell Biology of the Chromaffin Cell,Borges, R. and Gandia, L., Instituto Teofilo Hernando, , 2004
			Deoxyribonuclease I: 0.015%
			Hyaluronidase: 0.015%
大鼠	大鼠, SD, 雄性	Zona fasciculata/reticularis	Collagenase: 0.4%	Krebs-HEPES	Bruder Eric D, Ball Dennis L, Goodfriend Theodore L, Raff Hershel: An oxidized metabolite of linoleic acid stimulates corticosterone production by rat adrenal cells, Am J Physiol Regul Integr Comp Physiol 284, R1631-5, 2003
大鼠	大鼠, 威斯塔, 初生	Chromaffin cells	Collagenase Type 1: 0.025%	DMEM	Zhang L, Casl A, Avila E, Drucker-Col鉚n R, Escobar A:Immunocytochemical, ultrastructural and neurochemical evidences on synaptogenesis and dopamine release of rat chromaffin cells co-cultured with striatal neurons, J Neuropathol Exp Neurol 59, 170-4, 2000
大鼠	大鼠, 威斯塔, 初生	Chromaffin cells	Deoxyribonuclease I: 0.0015%	DMEM
大鼠	大鼠, SD, 雄性	ZG	Collagenase Type 1: 0.2%	Kreb's	Sayed, S., Whitehouse, B., and Jones, P.: Phosphoserine/Threonine Phosphatases in the Rat Adrenal Cortex: A Role in the Control of Steroidogenesis, J Endocrinol 154, 449, 1997
大鼠	大鼠, SD, 雄性	ZFR	Collagenase Type 1: 0.2%	Kreb's
大鼠	大鼠, 費希爾, 雄性, 10-16周	Adrenocortical	Deoxyribonuclease I: 0.005%	BSS	Roskelley, C.D. and Auersperg, N.: Density Separation of Rat Adrenocortical Cells: Morphology, Steroidogenesis, and P-450scc Expression in Primary Culture,In Vitro Cell Dev Biol 26, 493, 1990
大鼠	大鼠, 雄性, 120-160 g,	Leydig	Collagenase Type 2: 0.03% (adrenal)	Krebs Ringer bicarbonate buffer	Ng, T. and Liu, W.: Toxic Effect of Heavy Metals on Cells Isolated from the Rat Adrenal and Testis, In Vitro Cell Dev Biol 26, 24, 1990
大鼠	大鼠, SD, 雄性, 400-450 g	Adrenal	Collagenase Type 2: 0.03% (adrenal)	Krebs Ringer bicarbonate buffer
大鼠	大鼠, SD, 2-4 day old	Heart	Trypsin: 0.06%	25mM HEPES buffered Locke's solution, CMF	Trifaro, J., Tang, R., and Novas, M.: Monolayer Co-Culture of Rat Heart Cells and B綿羊 Adrenal Chromaffin Paraneurons, In Vitro Cell Dev Biol 26, 335, 1990
	(also 牛)	Adrenal chromaffin
		Paraneurons
大鼠	大鼠, Long-Evans, 雌性, 150-200 g	Glomerulosa	Collagenase: 0.2%	MEM-d-Val	Payet, N., Deziel, Y., and Lehoux, J.-G.: Vasopressin: A Potent Growth Factor in Adrenal Glomerulosa Cells in Culture, J Steroid Biochem 20, 449, 1984
大鼠	大鼠, 費希爾, 雄性, 1-10月	Adrenocortical	Deoxyribonuclease I: 0.005%	BSS	Leonard, R.K., Auersperg, N., and Parkes, C.O.: Ascorbic Acid Accumulation by Cultured Rat Adrenocortical Cells, In Vitro 19, 46, 1983
大鼠	大鼠, SD, 雄性, 400-450 克	Decapular	Deoxyribonuclease I: 0.01%	Medium 199	Li, C.H., Ng, T.B., and Cheng, C.H.K.: Melanotropins: Aldosterone- and Corticosterone-Stimulating Activity in Isolated Rat Adrenal Cells, Int J Pept Protein Res 19, 361, 1982
		Capsular
		Glomerulosa
大鼠	大鼠	Chromaffin	Trypsin: 0.10%	Ham's F-12 w/HEPES	Englert, D.F.: An Optical Study of Isolated Rat Adrenal Chromaffin Cells, Exp Cell Res 125, 369, 1980
大鼠	大鼠, 漢諾威-威斯塔, 第二出生后的一周 (還有豚鼠, 牛)	Chromaffin	Collagenase: 0.5%	HBSS	Unsicker, K., Rieffert, B., and Ziegler, W.: Effects of Cell Culture Conditions, Nerve Growth Factor, Dexamethasone, and Cyclic AMP on Adrenal Chromaffin Cells, Adv Biochem Psychopharmacol 25, 51, 1980
大鼠	大鼠 , SD, 雌性, 200 克	Glomerulosa	Deoxyribonuclease I: 0.05%	Kreb's	Braley, L., Williams, G., and Bradwin, G.: The Effect of Unit Gravity Sedimentation on Adrenal Steroidogenesis by Isolated Rat Glomerulosa and Fasciculata Cells, Endocrinology 106 (1), 50, 1980
大鼠	大鼠	Foreskin	Collagenase: 0.5%	Dulbecco's MEM w/10% calf serum	Folkman, 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	大鼠, 漢諾威-威斯塔, 7-12 天	Medullary	Trypsin: 0.125%	HBSS	Unsicker, K., Krisch, B., Otten, U., and Thoenen, H.: Nerve Growth Factor-Induced Fiber Outgrowth From Isolated Rat Adrenal Chromaffin Cells: Impairment by Glucocorticoids, Proc Natl Acad Sci U S A 75 (7), 3498, 1978
大鼠	大鼠, SD, 雄性	Cortical	Trypsin: 0.25%	Kreb's Ringer bicarbonate buffer	Barofsky, A., Feinstein, M., and Halkerston, I.: Enzymatic and Mechanical Requirements for the Dissociation of Cortical Cells From Rat Adrenal Glands,Exp Cell Res 79, 263, 1973
大鼠	大鼠, Holtzman, 雄性, 180-250 g	Adrenal	Collagenase Type 1: 0.5%	Kreb's Ringer bicarbonate buffer	Kloppenborg, P., Island, D., Liddle, G., Michelakis, A., and Nicholson, W.: A Method of Preparing Adrenal Cell Suspensions and Its Applicability to the In Vitro Study of Adrenal Metabolism, Endocrinology 82, 1053, 1968

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