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。

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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