肌组织消化手册

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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。

肌组织消化手册

物种	详细来源	细胞名称	消化用酶	培养基	参考文献
牛	牛	Muscle salite cells	Collagenase Type 2: 300 u/ml	Krebs-Ringer Bicarbonate	Lee, E., Choi, J., Hyun, J., Cho, K., Hwang, I., Lee, H., Chang, J. and Choi, I.: Steroid Effects on Cell Proliferation, Differentiation and Steroid Receptor Gene Expression in Adult Bovine Salite Cells., AJAS 20, 501, 2007
牛	牛	Pulmonary artery endothelial and smooth muscle cells	Collagenase: 0.04-0.05%	RPMI-1640	Yu, 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	牛	Smooth muscle	Trypsin: 0.25%	DMEM	Absher, M., Woodcock-Mitchell, J., Mitchell, J., Baldor, L., Low, R., and Warshaw, D.: Characterization of Vascular Smooth Muscle Cell Phenotype in Long-Term Culture, In Vitro Cell Dev Biol 25 (2), 183, 1989
牛	牛	Vascular smooth muscle	Elastase Type 3: 50 u/ml	PSS	Warshaw, D., Szarek, J., Hubbard, M., and Evans, J.: Pharmacology and Force Development of Single Freshly Isolated Bovine Cartoid Artery Smooth Muscle Cells,Circ Res 58, 399, 1986
牛	牛	Smooth muscle, fibroblasts	Trypsin: 0.055%	DMEM	Davies, P. and Kerr, C.: Modification of LDL Metabolism by Growth Factors in Cultured Vascular Cells and Human Skin Fibroblasts, Biochim Biophys Acta 712, 26, 1982
犬	犬	skeletal muscle cells	Collagenase Type 4: 200 u/ml	DMEM	Parker, M., Loretz, C., Tyler, A., Snider, L., Storb. R. and Tapscott, S.: Inhibition of CD26/DPP-IV Enhances Donor Muscle Cell Engraftment and Stimulates Sustained Donor Cell Proliferation., Skelet Muscle 2, 4, 2012
			Neutral Protease: 1 u/ml
犬	狗	Smooth muscle	Elastase: 50 u/ml	PSS	Subramanian, M., Madden, J., and Harder, D.: A Method for the Isolation of Cells from Arteries of Various Sizes, J Tiss Cul Meth 13, 13, 1991
犬	狗, 猎兔犬, 成年的	Smooth muscle	Elastase: 34 u/ml	Tyrode's solution w/ calcium	Wilde, D., and Lee, K.: Outward Potassium Currents in Freshly Isolated Smooth Muscle Cell of Dog Coronary Arteries, Circ Res 65, 1718, 1989
		Vascular
犬	狗	Artery	Elastase: 80 u/ml	PSS	Dobrin, P., and Canfield, T.: Elastase, Collagenase, and the Biaxial Elastic Properties of Dog Carotid Artery, Am J Physiol Heart Circ Physiol 247 (16), H124, 1984
		Carotid
鸡	鸡, 1-2 天	Gizzard and aorta smooth muscle	Collagenase Type 1: 0.15%	HBSS	Dirksen W., Vladic F., and Fisher S.: A Myosin Phosphatase Targeting Subunit Isoform Transition Defines a Smooth Muscle Developmental Phenotypic Switch, Am J Physiol/Cell 278(3), C589, 2000
鸡	小鸡	Smooth muscle	Trypsin: 0.05% - 0.1%	HBSS	Chamley-Campbell, J., Campbell, G., and Ross, R.: The Smooth Muscle in Cell Culture, Physiol Res 59, 1, 1979
鸡	小鸡,白来杭鸡, 12 天	Muscle	Trypsin: 0.25%	Puck's saline A	Bullaro, J., and Brookman, D.: Comparison of Skeletal Muscle Monolayer Cultures Initiated With Cells Dissociated by the Vortex and Trypsin Methods, In Vitro 12, 564, 1976
鸡	小鸡, 白来杭鸡, 胚胎, 11 天	Muscle	Trypsin: 0.05%	Saline G	Tepperman, K., Morris, G., Essien, F., and Heywood, S.M.: A Mechanical Dissociation Method For Preparation of Muscle Cell Cultures, J Cell Physiol 86, 561, 1975
鸡	小鸡胚胎	Thyroid	Collagenase: 0.25%	Tyrode's saline, potassium free	Hilfer, S., and Brown, J.: Collagenase. Its Effectiveness as a Dispersing Agent for Embryonic Chick Thyroid and Heart, Exp Cell Res 65, 246, 1971
		Muscle
		Heart
鸡	小鸡, 胚胎	Muscle	Trypsin: 0.1%	CMF HBSS	Hilfer, S.: Collagenase Treatment of Chick Heart and Thyroid,Tissue Cult Methods & Applications,Kruse, P., and Patterson, M., , 246, 1971
鸡	小鸡胚胎	Various tissues (heart, liver, skeletal, cardiac)	Trypsin: various grades	CMF Tyrode's solution	Rinaldini, L.: An Improved Method for the Isolation and Quantitative C*tion of Embryonic Cells, Exp Cell Res 16, 477, 1959
猫科动物	猫, 成年杂种, 任一性别, 2.5-4.0 公斤	Cerebral arteries	Elastase: 50 u/ml	Puck's solution	Madden, J., Vadula, M., and Kurup, V.: Effects of Hypoxia and Other Vasoactive Agents on Pulmonary and Cerebral Artery Smooth Muscle Cells, Am J Physiol 263, L384, 1992
猫科动物	猫, 杂交的, 成年的, 2-4 公斤	Myocytes	Collagenase: 0.12%	Kreb's Henseleit, CF	Follmer, C.H., Ten Eick, R.E., and Yeh, J.Z.: Sodium Current Kinetics in Cat Atrial Myocytes, J Physiol 384, 169, 1987
鱼	角鲨,海绿角鲨 , 2-6 公斤	Myocytes, heart and stomach	Protease XIV: 0.028%	Solution C	Mitra, R. and Morad, M.: A Uniform Enzymatic Method for Dissociation of Myocytes from Hearts and Stomachs of Vertebrates, Am J Physiol 249, H1056, 1985
	(包括豚鼠，兔, 青蛙)			(see reference)
青蛙	青蛙, 有爪蟾蜍, 胚胎期 17 & 19	Muscle	Collagenase: 0.10%	Steinberg's solution	Stollberg, J. and Fraser, S.: Acetylcholine Receptors and Con A-Binding Sites onXenopus Muscle Cells, J Cell Biol 107, 1397, 1988
青蛙	青蛙	Myocytes	Trypsin: 0.1%	CF Ringer	Shepherd, N. and Kavaler, F.: Direct Control of Contraction Force of Single Frog Atrial Cells by Extracellular Ions, Am J Physiol 251, C653, 1986
青蛙	青蛙, 美洲豹蛙(50-100 克)	Myocytes, heart and stomach	Protease XIV: 0.028%	Solution C	Mitra, R. and Morad, M.: A Uniform Enzymatic Method for Dissociation of Myocytes from Hearts and Stomachs of Vertebrates, Am J Physiol 249, H1056, 1985
	(包括豚鼠, 兔, 角鲨,海绿角鲨)			(see reference)
青蛙	青蛙, 有爪蟾蜍	Muscle	Trypsin: 0.5%	L15 medium	Anderson, M.J., Cohen, M.W., and Zorychta, E.: Effects of Innervation on the Distribution of Acetylcholine Receptors on Cultured Muscle Cells, J Physiol 268, 731, 1977
				(see reference)
豚鼠	豚鼠	Bladder smooth muscle	Collagenase Type 2: 0.1-0.2%	Krebs-Ringer bicarbonate	Shieh CC, Feng J, Buckner SA, Brioni JD, Coghlan MJ, Sullivan JP, Gopalakrishnan M: Functional implication of spare ATP-sensitive K(+) channels in bladder smooth muscle cells, J Pharmacol Exp Ther 296, 669-75, 2001
豚鼠	豚鼠, 200-380 克	Capillaries	Collagenase Type 2: 0.15%	CF solution	Schnitzler, M., Derst, C., Daut, J., and Preisig-Muller, R.: ATP-Sensitive Potassium Channels in Capillaries Isolated From Guinea-Pig Heart, J Physiol 525 (2), 307, 2000
		Myocytes
豚鼠	豚鼠, 2-4 周大, 雄性, 雌性	Smooth muscle	Papain: 0.1%	Krebs solution	Firth, T., Mawe, G., and Nelson, M.: Pharmacology and Modulation of K_ATPChannels by Protein Kinase C and Phosphatates in Gallbladder Smooth Muscle, Am J Physiol Cell Physiol 278, C1031, 2000
		Gallbladder
豚鼠	豚鼠, 成年的, 250-350 克	Smooth muscle	Papain: 0.1%	NaCl, sodium glutamate, MgCl, KCl, glucose, Kreb's, and HEPES	Jennings, L., Xu, Q., Firth, T., Nelson, M., and Mawe, G.: Cholesterol Inhibits Spontaneous Action Potentials and Calcium Currents in Guinea Pig Gallbladder Smooth Muscle, Am J Physiol 277, G1017, 1999
		Gallbladder
豚鼠	Dunkin-Hartley豚鼠, 雌性	Myocytes	Protease:	DMEM	Ryder, K., Bryant, S., and Hart, G.: Membrane Current Changes in Left Ventricular Myocytes Isolated From Guinea-Pigs After Abdominal Aortic Coarctation, Cardiovasc Res 27, 1278, 1993
豚鼠	豚鼠	Smooth muscle	Trypsin: 0.1%	Potassium buffer solution	Hu, S., and Kim, H.: Activation of K+ Channel in Vascular Smooth Muscle by Cytochrome P-450 Metabolites of Arachidonic Acid, FASEB J 6, A383, 1992
	(包括大鼠, 兔)
豚鼠	豚鼠, 200-300 克	Smooth muscle	Collagenase: 0.3%	CF solution	Ohya, Y. and Sperelakis, N.: ATP Regulation of the Slow Calcium Channels in Vascular Smooth Muscle Cells, Circ Res 64, 145, 1989
		Mesenteric artery
豚鼠	豚鼠, 200-400克	Myocytes, heart and stomach	Protease XIV: 0.028%	Solution C	Mitra, R. and Morad, M.: A Uniform Enzymatic Method for Dissociation of Myocytes from Hearts and Stomachs of Vertebrates, Am J Physiol 249, H1056, 1985
	(包括兔, 青蛙, 角鲨)			(see reference)
豚鼠	豚鼠, 青春期前的	Smooth muscle	Trypsin: 0.05%	Dulbecco-Vogt modification of Eagle's	Ross, R.: The Smooth Muscle Cell . II. Growth of Smooth Muscle in Culture and Formation of Elastic Fibers, J Cell Biol 50, 172, 1971
		Aortic
仓鼠	仓鼠, 雄性, 60-70 天	Salite	Trypsin: 0.25%	DMEM	Nakamura, T., Iwata, Y., Sampaolesi, M., Hanada, H., Saito, N., Artman, M., Coetzee, W., and Shigekawa, M.: Stretch-Activated Cation Channels in Skeletal Muscle Myotubes From Sarcoglycan-Deficient Hamsters, Am J Physiol Cell Physiol281, C690, 2001
人类	人类	Skeletal muscle stem cells	Collagenase Type 2: 750 u/ml	Ham's F10	Charville, G., Cheung, T., Yoo, B., Santos, P., Lee, G., Shrager, J. and Rando, T.:Ex Vivo Expansion and In Vivo Self-Renewal of Human Muscle Stem Cells.,Stem Cell Reports Vol. 5,, , 621, 2015
			Neutral Protease: 2 u/ml
人类	人类	Smooth muscle	Collagenase Type 1: 0.1%	DMEM	Lu, S., Sun, X., Hong, T., Song, K., Yang, S. and Wang, C.: Isolation and Culture of Smooth Muscle Cells from Human Acute Type A Aortic Dissection., J Cardiothorac Surg 8, 83, 2013
人类	人类, 男性	Myogenic	Collagenase Type 4: 0.1%	HBSS	Stadler, G., Chen, J., Wagner, K., Robin, J., Shay, J., Emerson, C. and Wright, W.: Establishment of Clonal Myogenic Cell Lines from Severely Affected Dystrophic Muscles - CDK4 Maintains the Myogenic Population., Skelet Muscle 1, 12, 2011
			Neutral Protease: 2.4 u/ml
人类	人类	Muscle derived multiprogenitor cells	Collagenase Type 2: 0.05%	DMEM	Nesti, L., Jackson, W., Shanti, R., Koehler, S., Aragon, A., Bailey, J., Sracic, M., Freedman, B., Giuliani, J. and Tuan, R.: Differentiation Potential of Multipotent Progenitor Cells Derived from War-Traumatized Muscle Tissue., J Bone Joint Surg Am90, 2390, 2008
人类	人类	Endothelial and vascular smooth muscle	Collagenase Type 1: 0.2%	HBSS	Moss, 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	Urinary tract smooth muscle	Collagenase Type 4: 100 u/ml	DMEM	Kimuli, M., Eardley, I. and Southgate, J.: In Vitro Assessment of Decellularized Porcine Dermis as a Matrix for Urinary Tract Reconstruction., BJU Int 94, 859, 2004
人类	人类, 女性	Smooth muscle	Deoxyribonuclease I: 0.015% and 0.007%	HBSS	Richardson, M., Taylor, D., Casey, M., MacDonald, P., and Stull, J.: Biochemical Markers of Contraction in Human Myometrial Smooth Muscle Cells in Culture, In Vitro Cell Dev Biol 23, 21, 1987
		Myometrial
人类	人类, 女性	Smooth muscle	Deoxyribonuclease I: 0.12%	HBSS	Casey, M., MacDonald, P., Mitchell, M., and Snyder, J.: Maintenance and Characterization of Human Myometrial Smooth Muscle Cells in Monolayer Culture, In Vitro 20, 396, 1984
		Myometrial
人类	人类, 胎儿	Smooth muscle, fibroblasts	Trypsin: 0.055%	DMEM	Davies, P. and Kerr, C.: Modification of LDL Metabolism by Growth Factors in Cultured Vascular Cells and Human Skin Fibroblasts, Biochim Biophys Acta 712, 26, 1982
	(包括牛)
人类	人类	Smooth muscle	Trypsin: 0.25%	DMEM	Eskin, S., Sybers, H., Lester, J., Navarro, L., Gotto, A., and DeBakey, M.: Human Smooth Muscle Cells Cultured From Atherosclerotic Plaques and Uninvolved Vessel Wall, In Vitro 17 (8), 713, 1981
人类	人类	Smooth muscle	Trypsin: 0.05% - 0.1%	HBSS	Chamley-Campbell, J., Campbell, G., and Ross, R.: The Smooth Muscle in Cell Culture, Physiol Res 59, 1, 1979
	(包括大鼠, 豚鼠, 小鸡, 猴)
蜥蜴	蜥蜴(安乐蜥)	Myoblasts, tail	Collagenase: 0.2%	GM III	Cox, P., and Simpson, Jr., S.: A Microphotometric STudy of Myogenic Lizard Cells Grown in Vitro, Dev Biol 23, 433, 1970
				(see reference)
猴	猴 (豚尾猴)	Smooth muscle	Trypsin: 0.05%	Dulbecco-Vogt	Chait, A., Ross, R., Albers, J., and Bierman, E.: Plaet-Derived Growth Factor Stimulates Activity of LDL Receptors, Proc Natl Acad Sci U S A 77, 4084, 1980
猴	猕猴, 1 岁	Smooth muscle, saphenous vein	Elastase: 0.05%	BSS	Chamley, J., Campbell, G., McConnell, J., and Groschel-Stewart, U.: Comparison of Vascular Smooth Muscle Cells from Adult Human, Monkey and Rabbit in Primary Culture and in Subculture, Cell Tissue Res 177, 503, 1977
	(包括人类, 兔)
小鼠	小鼠, 6-8 周	Myofibers	Collagenase Type 1: 0.2%	DMEM	Pasut, A., Jones, A. and Rudnicki, M.: Isolation and Culture of Individual Myofibers and their Salite Cells from Adult Skeletal Muscle., J Vis Exp 73, e50074, 2013
小鼠	小鼠	Vascular Smooth Muscle	Collagenase Type 2: 0.14%	Basal Culture	Metz, R., Patterson, J. and Wilson, E.: Vascular Smooth Muscle Cells: Isolation, Culture, and Characterization., Methods Mol Biol 843, 169, 2012
小鼠	小鼠, 10 周	Disphragm cells	Collagenase Type 2: 100 u/ml	PBS	Rezk, B., Yoshida, T., Semprun-Prieto, L., Higashi, Y., Sukhanov, S. and Delafontaine, P.: Angiotensin II Infusion Induces Marked Diaphragmatic Skeletal Muscle Atrophy., PLoS ONE 7, e30276, 2012
			Pronase: 0.125%
小鼠	小鼠	Skeletal muscle fiber	Collagenase Type 1: 0.2%	Tyrode	Weisleder, N., Zhou, J. and Ma, J.: Detection of Calcium Sparks in Intact and Permeabilized Skeletal Muscle Fibers., Methods Mol Biol 798, 395, 2012
小鼠	小鼠	Myoblast	Collagenase Type 2: 0.2%	DMEM	Shi, H., Boadu, E., Mercan, F., Le, A., Roth Flach, R., Zhang, L., Tyner, K., Olwin, B. and Bennett, A.: MAP Kinase Phosphatase-1 Deficiency Impairs Skeletal Muscle Regeneration and Exacerbates Muscular Dystrophy., FASEB J 24, 2985, 2010
小鼠	小鼠, 1-25 个月	Myocytes, endothelial	Neutral Protease: 1.2 u/ml	PBS	Ieronimakis Nicholas, Balasundaram Gayathri, Reyes Morayma: Direct isolation, culture and transplant of mouse skeletal muscle derived endothelial cells with angiogenic potential, PLoS ONE 3, e0001753, 2008
			Collagenase Type 4: 0.2%
小鼠	小鼠, 15-20 天	Intersitial cells of Cajal	Collagenase Type 2: 0.13%	M199	Li CX, Liu BH, Tong WD, Zhang LY, and Jiang YP: Dissociation, culture and morphologic changes of interstitial cells of Cajal in vitro, World J Gastroenterol 11, 2838, 2005
小鼠	小鼠,新生的	Skeletal muscle myotubes	NCIS kit: per instructions	L-15	Johnson, B., Scheuer, T. and Catterall, W.: Convergent Regulation of Skeletal Muscle Ca2+ Channels by Dystrophin, the Actin Cytoskeleton, and cAMP-Dependent Protein Kinase., Proc Natl Acad Sci U S A 102, 4191, 2005
小鼠	小鼠, 雄性, 6-14 周	Precursor cells	Collagenase Type 2: 0.5%	DMEM/F12	Winitsky, S., Gopal, T., Hassanzadeh, S., Takahashi, H., Gryder, D., Rogawski, M., Takeda, K., Yu, Z., Xu, Y. and Epstein, N.: Adult Murine Skeletal Muscle Contains Cells That Can Differentiate into Beating Cardiomyocytes In Vitro., PLoS Biol3, e87, 2005
小鼠	小鼠, 6-8 周	Skeletal muscle progenitor	Collagenase Type 2: 0.2%	DMEM	Majka S., Jackson K., Kienstra K., Majesky M., Goodell M., and Hirschi K.: Distinct Progenitor Populations in Skeletal Muscle Are Bone Marrow Derived and Exhibit Different Cell Fates During Vascular Regeneration, J Clin Invest 111, 71, 2003
小鼠	小鼠, neonatal	Myocytes	Collagenase Type 1: 0.5%	DMEM	Fukada S., Miyagoe-Suzuki Y., Tsukihara H., Yuasa K., Higuchi S., Ono S., Tsujikawa K., Takeda S., and Yamamoto H.: Muscle Regeneration by Reconstitution with Bone Marrow or Fetal Liver Cells from Green Fluorescent Protein-gene Transgenic Mice, J Cell Sci 115, 1285, 2002
小鼠	小鼠, 6-8 week	Myocytes	Collagenase Type 2: 0.2%	HBSS	McKinney-Freeman SL, Jackson KA, Camargo FD, Ferrari G, Mavilio F, Goodell MA.: Muscle-derived hematopoietic stem cells are hematopoietic in origin, Proc Natl Acad Sci U S A 99, 1341, 2002
			Trypsin: 0.25%
绵羊	绵羊, 成年和新生的	Tracheal smooth muscle cells	Papain: 0.2%	MOPS-PSS	Driska S., Laudadio R., Wolfson M., and Shaffer T.: A Method for Isolating Adult and Neonatal Airway Smooth Muscle Cells and Measuring Shortening Velocity, J Appl Physiol 86(1), 427, 1999
			Deoxyribonuclease I: 0.1%
猪	猪, 2个月	Skeletal muscle	Collagenase: 10%	HBSS	Lewis, F., Henning, B., Marazzi, G., Sassoon, D., Ellison, G. and Nadal-Ginard, B.: Porcine Skeletal Muscle-Derived Multipotent PW1pos/Pax7neg Interstitial Cells: Isolation, Characterization, and Long-Term Culture., Stem Cells Transl Med 3, 702-12, 2014
			Neutral Protease: 0.3%
猪	猪, 雄性, 25-40 公斤	Coronary myocytes	Collagenase Type 2: 294 u/ml	low calcium physiological saline	Korzick, D., Laughlin, M., and Bowles, D.: Alterations in PKC signaling underlie enhanced myogenic tone in exercise-trained porcine coronary resistance arteries, J Appl Physiol 96, 1425-32, 2004
			Elastase: 6.5 u/ml
			Deoxyribonuclease I: 0.04%
			Soybean Trypsin Inhibitor: 0.1%
猪	猪, 成年的, 35-45 公斤	Arterial smooth muscle	Collagenase Type 2: 294 u/ml	MEM	Wamhoff BR, Dixon JL, Sturek M: Atorvastatin treatment prevents alterations in coronary smooth muscle nuclear Ca2+ signaling in diabetic dyslipidemia, J Vasc Res 39, 208, 2002
			Elastase: 6.5 u/ml
			Deoxyribonuclease I: 0.4 mg/ml
			Soybean Trypsin Inhibitor: 1 mg/ml
猪	约克夏猪, 30公斤	Coronary smooth muscle	Collagenase Type 2: 150 u/ml	HBSS	Sirous ZN, Fleming JB, Khalil RA: Endothelin-1 enhances eicosanoids-induced coronary smooth muscle contraction by activating specific protein kinase C isoforms, Hypertension 37, 497-504, 2001
			Elastase: 0.05%
猪	猪	Bladder smooth muscle	Collagenase Type 2: 0.1-0.2%	Krebs-Ringer bicarbonate	Shieh CC, Feng J, Buckner SA, Brioni JD, Coghlan MJ, Sullivan JP, Gopalakrishnan M: Functional implication of spare ATP-sensitive K(+) channels in bladder smooth muscle cells, J Pharmacol Exp Ther 296, 669-75, 2001
猪	猪, 少年的, 30公斤	Coronary smooth muscle cells	Collagenase: 0.3%	HBSS	Huckle WR, Drag MD, Acker WR, Powers M, McFall RC, Holder DJ, Fujita T, Stabilito II, Kim D, Ondeyka DL, Mantlo NB, Chang RS, Reilly CF, Schwartz RS, Greenlee WJ, Johnson RG: Effects of subtype-selective and balanced angiotensin II receptor antagonists in a porcine coronary artery model of vascular restenosis,Circulation 93, 1009-19, 1996
			Elastase: 0.05%
猪	猪	Smooth muscle, aorta	Collagenase: 0.3%	DMEM	Xiong, Yimin, Xu, Shangzhe, and Slakey, Linda L: Modulation of Response to Adenosine in Vascular Smooth Muscle Cells Cultured in Defined Medium, In Vitro Cell Dev Biol 27, 355, 1991
猪	猪	Smooth muscle	Trypsin: 0.05%	EDTA 0.02%	Breton, M., Berrou, E., Deudon, E. and Picard, J.: Changes in Proteoglycans of Cultured Pig Aortic Smooth Muscle Cells During Subculture, In Vitro Cell Dev Biol26, 157, 1990
		Aorta
猪	猪	Smooth muscle	Collagenase: 0.30%	DMEM	Fehr, T., Dickinson, E., Goldman, S. and Slakey, L.: Cyclic AMP Efflux is Regulated by Occupancy of The Adenosine Receptor in Pig Aortic Smooth Muscle Cells, J Biol Chem 265, 10974, 1990
		Aortic medial tissue
鹌鹑	鹌鹑, 胚胎, 10 天	Myoblasts	Collagenase Type 2: 0.1%	Puck's solution	Konigsberg, I.: Skeletal Myoblasts in Culture, Methods Enzymol LVIII, 511, 1979
兔	兔,新西兰白	Aortic smooth muscle	Collagenase Type 2: 300 u/ml	F10 Ham's	Croons Valerie, Martinet Wim, Herman Arnold G, Timmermans Jean-Pierre, De Meyer Guido R Y: Selective clearance of macrophages in atherosclerotic plaques by the protein synthesis inhibitor cycloheximide, J Pharmacol Exp Ther 320, 986-93, 2007
			Elastase: 5 u/ml
兔	兔, 新西兰, 雄性, 2.5-3.5 公斤	Enterocytes	Trypsin: 0.1%	RPMI 1640 w/ 1% fetal bovine serum	Santos, M., Nguyen, B.., Thompson, J.: Factors Affecting in Vitro Growth of Harvested Enterocytes, Cell Transplant 1, 299, 1992
				PBS
兔	兔	Smooth muscle	Trypsin: 0.1%	Potassium buffer solution	Hu, S., and Kim, H.: Activation of K+ Channel in Vascular Smooth Muscle by Cytochrome P-450 Metabolites of Arachidonic Acid, FASEB J 6, A383, 1992
	(包括大鼠, 豚鼠)
兔	兔,成年的, 1-2 公斤	Smooth muscle	Elastase: 0.17 - 0.25%	Saline	Benham, C., Bolton, T., Byrne, N., and Large, W.: Action of Extremely Applied Adenosine Triphosphate O Single Smooth Muscle Cells Dispersed From Rabbit Ear Artery, J Physiol 387, 473, 1987
兔	兔, 新西兰白, 1500 克	Smooth muscle, aortic	Trypsin: 0.038%	MEM	Knodle, S., Anderson, S., and Papaioannou, S.: Large Scale Preparation of Rabbit Aortic Smooth Muscle Cells For Use in Calcium Uptake Studies, In Vitro Cell Dev Biol 22, 23, 1986
兔	兔, 成年的, 1-2 公斤	Smooth muscle, ear artery	Trypsin: 0.1%	CF solution	Benham, C.D., Bolton, T.B.: Spontaneous Transient Outward Currents in Single Visceral and Vascular Smooth Muscle Cells of the Rabbit, J Physiol 381, 385, 1986
				(see reference)
兔	兔, 0.5-1 公斤	Myocytes, heart and stomach	Protease XIV: 0.028%	Solution C	Mitra, R. and Morad, M.: A Uniform Enzymatic Method for Dissociation of Myocytes from Hearts and Stomachs of Vertebrates, Am J Physiol 249, H1056, 1985
	(包括豚鼠, 青蛙, 角鲨)			(see reference)
兔	兔, 新西兰白, 成年的, 雄性, 2 公斤	Smooth muscle, aorta	Trypsin: 0.1%	Krebs Ringer HEPES solution	Ives, H., Schultz, G., Galardy, R., and Jamieson, J.: Preparation of Functional Smooth Muscle Cells from the Rabbit Aorta, J Exp Med 148, 1400, 1978
兔	兔, 新西兰白白化, 5-6 个月	Smooth muscle, saphenous vein	Elastase: 0.05%	BSS	Chamley, J., Campbell, G., McConnell, J., and Groschel-Stewart, U.: Comparison of Vascular Smooth Muscle Cells from Adult Human, Monkey and Rabbit in Primary Culture and in Subculture, Cell Tissue Res 177, 503, 1977
	(包括人类, 猕猴, 兔)
兔	兔, 南美洲栗鼠, 5-8 月, 处女, 雌性, 2-3 公斤	Smooth muscle, aorta	Hyaluronidase: 800 u/ml	HBSS	Peters, T., Muller, M., and deDuve, C.: Lysosomes of the Arterial Wall. I. Isolation and Subcellular Fractionation of Cells from Normal Rabbit Aorta, J Exp Med 136, 1117, 1972
兔	兔, 新英格兰, 白化变种	Thoracic aorta	Elastase: 0.008%	Kreb's Ringer	天, A., Phil, D., and Newman, H.: Synthesis of Phospholipid by Foam Cells Isolated from Rabbit Atherosclerotic Lesions, Circ Res XIX, 122, 1966
大鼠	SD大鼠,	Pulmonary arterial smooth muscle	Collagenase Type 2: 0.1%	Buffer	Zeng, Y., Liu, H., Kang, K., Wang, Z., Hui, G., Zhang, X., Zhong, J., Peng, W., Ramchandran, R., Raj, J. and Gou, D.: Hypoxia Inducible Factor-1 Mediates Expression of miR-322: Potential Role in Proliferation and Migration of Pulmonary Arterial Smooth Muscle Cells., Sci Rep 5, 12098, 2015
			Elastase: 0.05%
大鼠	大鼠	Skeletal muscle fiber	Collagenase Type 1: 0.2%	Tyrode	Weisleder, N., Zhou, J. and Ma, J.: Detection of Calcium Sparks in Intact and Permeabilized Skeletal Muscle Fibers., Methods Mol Biol 798, 395, 2012
大鼠	大鼠, 胎儿 17-18 天	Myotubes	Trypsin: 0.05%	see reference	Das, M., Rumsey, J., Bhargava, N., Stancescu, M. and Hickman, J.: Skeletal Muscle Tissue Engineering: A Maturation Model Promoting Long-Term Survival of Myotubes, Structural Development of the Excitation-Contraction Coupling Apparatus and Neonatal Myosin Heavy Chain Expression., Biomaterials 30, 5392, 2009
大鼠	大鼠	Myogenic	Collagenase Type 2: 1.0%	Ham's F-10	Pacak, C. and Cowan, D.: Fabrication of Myogenic Engineered Tissue Constructs., J Vis Exp 27, e1137, 2009
			Neutral Protease: 2.4 u/ml
大鼠	Lewis大鼠, 初生的	Myoblasts	Collagenase Type 2: 1.0%	Ham's F-10	Kim, J., Hadlock, T., Cheney, M., Varvares, M. and Marler, J.: Muscle Tissue Engineering for Partial Glossectomy Defects., Arch Facial Plast Surg 5, 403, 2003
			Neutral Protease: 2.4 u/ml
大鼠	SD大鼠, 成年和出生一天的	Myooids	Neutral Protease: 4 u/ml	Ham's F-12	Dennis, R., Kosnik II, P., Gilbert, M., and Faulkner, J.: Excitability and Contractility of Skeletal Muscle Engineered from Primary Cultures and Cell Lines, Am J Physiol Cell Physiol 280, C288, 2001
大鼠	SD大鼠, 3 个月	Smooth muscle cells	Collagenase Type 2: 0.2%	M-199	Su E., Stevenson S., Rollence M., Marshall-Neff J., and Liau G.: A Genetically Modified Adenoviral Vector Exhibits Enhanced Gene Transfer of Human Smooth Muscle Cells, J Vasc Res 38(5), 471, 2001
			Elastase: 0.04%
			Soybean Trypsin Inhibitor: 0.1%
大鼠	SD大鼠, 250 克	Arterial smooth muscle	Papain: 0.03%	see reference	Jaggar JH: Intravascular pressure regulates local and global Ca(2+) signaling in cerebral artery smooth muscle cells, Am J Physiol Cell Physiol 281, C439-48, 2001
			Collagenase: 0.1%
大鼠	Wistar大鼠, 成年的, 雄性	Smooth & skeletal muscle	Protease: 0.01%	PSS	Wellman, G., Barrett-Jolley, R., Koppel, H. Everitt, D., and Quayle, J.: Inhibition of Vascular K_ATP Channels by U-37883A: A Comparison with Cardiac and Skeletal Muscle, Br J Pharmacol 128, 909, 1999
		Cardiac myocytes
大鼠	SD大鼠, 雄性, 250-350 克	Vascular smooth muscle	Collagenase Type 2: 0.1%	DMEM	Hrometz, S., Edelmann, S., McCune, D., Olges, J., Hadley, R., Perez ,D., and Piascik, M.: Expression of Multiple Alpha1-Adrenoceptors on Vascular Smooth Muscle: Correlation with the Regulation of Contraction, J Pharmacol Exp Ther 290(1), 452, 1999
			Elastase: 0.0125%
大鼠	SD大鼠, 雄性, 150-175 克	Smooth muscle, endothelial	Trypsin: 0.04%	MEM	Redmond, E., Cahill, P., and Sitzmann, J.: Perfused Transcapillary Smooth Muscle and Endothelial Cell Co-Culture-A Novel In Vitro Model, In Vitro Cell Dev Biol Anim 31, 601, 1995
大鼠	Wistar京都大鼠, 10-15 周	Smooth muscle, mesenteric artery	Trypsin: 0.05%	MEM	McGuire, P., Walker-Caprioglio, H., Little, S., and McGuffee, L.: Isolation and Culture of Rat Superior Mesenteric Artery Smooth Muscle Cells, In Vitro Cell Dev Biol 29, 135, 1993
大鼠	大鼠	Smooth muscle	Trypsin: 0.1%	Potassium buffer solution	Hu, S., and Kim, H.: Activation of K+ Channel in Vascular Smooth Muscle by Cytochrome P-450 Metabolites of Arachidonic Acid, FASEB J 6, A383, 1992
	(包括兔, 豚鼠)
大鼠	SD大鼠, 19 天	Smooth muscle, myometrial	Trypsin: 150 礸/ml	HBSS or PSS, CMF	Loch-Caruso, R., Pahl, M., and Juberg, D.: Rat Myometrial Smooth Muscle Cells Show High Levels of Gap Junctional Communication Under a Variety of Culture Conditions, In Vitro Cell Dev Biol 28, 97, 1992
大鼠	SHRs 和 WKY大鼠, 雄性, 10 - 14 周	Smooth muscle, tail arteries	Papain: 0.1%	HEPES buffer	Bolzon, B. and Cheung, D.: Isolation and Characterization of Single Vascular Smooth Muscle Cells From Spontaneously Hypertensive Rats, Hypertension 14, 137, 1989
				(see reference)
大鼠	大鼠, 1-3 天	Smooth muscle, aortic	Elastase: 0.0125%	DMEM	Barone, L., Wolfe, L., Faris, B., and Franzblau, C.: Elastin mRNA Levels and Insoluble Elastin Accumulation in Neonatal Rat, Biochemistry 27, 3175, 1988
大鼠	大鼠	Muscle, mesenteric arteries	Trypsin: 0.05%	HEPES KG solution	Bean, B., Sturek, M., Puga, A., and Hermsmeyer, K.: Calcium Channels in Muscle Cells Isolated From Rat Mesenteric Arteries: Modulation by Dihydropyridine Drugs,Circ Res 59, 229, 1986
				(see reference)
大鼠	SHR和WKY大鼠, 任一性别, 12-19 天, 3 个月, 和退休饲养者	Endothelial, aortic	Elastase: 0.05%	Waymouth's culture medium	Gordon, D., Mohai, L., and Schwartz, S.: Induction of Polyploidy in Cultures of Neonatal Rat Aortic Smooth Muscle Cells, Circ Res 59, 633, 1986
大鼠	大鼠, 雄性, 150-250 克	Endothelial, aortic	Trypsin:	RPMI 1640	Cole, O., Fan, T., and Lewis, G.: Isolation, Characterization, Growth and Culture of Endothelial Cells From the Rat Aorta, Cell Biol Int Rep 10 (6), 399, 1986
大鼠	Wistar大鼠, 雌性, 10 周	Myocytes	Collagenase: 0.1%	HBSS	Boulanger-Saunier, C., Kattenburg, D., and Stoclet, J.: Cyclic AMP-dependent Phosphorylation of a 16kDa Protein in a Plasma Membrane-enriched Fraction of Rat Aortic Myocytes, F.E.B.S. Lett. 193, 283, 1985
大鼠	SD大鼠, 雄性, 200 - 250 克	Smooth muscle, thoracic aorta	Trypsin: 0.0375%	Eagle's MEM with calcium	Brock, T., Alexander, R., Ekstein, L., Atkinson, W., and Gimbrone, M.: Angiotensin Increases Cytosolic Free Calcium in Cultured Vascular Smooth Muscle Cells,Hypertension 7, 105, 1985
大鼠	SD大鼠, 225-250克	Mesenteric artery smooth muscle cells	Elastase: .0125%	HBSS	Gunther S, Alexander RW, Atkinson WJ, and Gimbrone MA Jr.: Functional angiotensin II receptors in cultured vascular smooth muscle cells, J Cell Biol 92, 289, 1982
			Soybean Trypsin Inhibitor: 0.025%
			Collagenase Type 1: 0.1%
大鼠	大鼠, 3-4 天	Myocardial	Trypsin NF 1:250: 0.125%	HBSS CMF	Kasten, F.: Rat Myocardial Cells In Vitro: Mitosis and Differentiated Properties, In Vitro 8, 128, 1972
大鼠	大鼠, 200 克	Muscle	Trypsin: 0.05%	Kreb's Henseleit bicarbonate buffer	Kono, T.: Roles of Collagenases and Other Proteolytic Enzymes in the Dispersal of Animal Tissues, Biochim Biophys Acta 178, 397, 1969
大鼠	Wistar大鼠, 3-10 天	Heart	Trypsin NF 1:250: 250: 0.1%	Saline A	Harary, I., and Farley, B.: In Vitro Studies on Single Beating Rat Heart Cells, Exp Cell Res 29, 451, 1963
				(See reference)

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