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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。
肌組織消化手冊
| 物種 | 詳細來源 | 細胞名稱 | 消化用酶 | 培養基 | 參考文獻 |
| 牛 | 牛 | 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, 獵兔犬, 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 | |||||
| 雞 | 雞, 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 |
| 雞 | 小雞 | 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 |
| 雞 | 小雞,白來杭雞, 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 |
| 雞 | 小雞, 白來杭雞, 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 |
| 雞 | 小雞 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 | |||||
| 雞 | 小雞, 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 |
| 雞 | 小雞 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 |
| 貓科動物 | 貓, 成年雜種, 任一性別, 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 |
| 貓科動物 | 貓, 雜交的, 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 |
| 魚 | 角鯊,海綠角鯊 , 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, 角鯊,海綠角鯊) | (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 KATPChannels 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, 角鯊) | (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 | |||||
| 倉鼠 | 倉鼠, 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 |
| 人類 | 人類 | 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 | |||||
| 人類 | 人類 | 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 |
| 人類 | 人類, 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 | |||||
| 人類 | 人類 | 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 |
| 人類 | 人類 | 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 |
| 人類 | 人類 | 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 |
| 人類 | 人類, 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 | |||||
| 人類 | 人類, 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 | |||||
| 人類 | 人類, 胎兒 | 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) | |||||
| 人類 | 人類 | 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 |
| 人類 | 人類 | 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, 小雞, 猴) | |||||
| 蜥蜴 | 蜥蜴(安樂蜥) | 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 | 獼猴, 1 歲 | 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 人類, 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 個月 | 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% | |||||
| 綿羊 | 綿羊, 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% | |||||
| 豬 | 豬, 2個月 | 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% | |||||
| 豬 | 豬, 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% | |||||
| 豬 | 豬, 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 | |||||
| 豬 | 約克夏豬, 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% | |||||
| 豬 | 豬 | 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 |
| 豬 | 豬, 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% | |||||
| 豬 | 豬 | 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 |
| 豬 | 豬 | 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 | |||||
| 豬 | 豬 | 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 | |||||
| 鵪鶉 | 鵪鶉, 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,新西蘭白 | 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, 新西蘭, 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, 新西蘭白, 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, 角鯊) | (see reference) | ||||
| 兔 | 兔, 新西蘭白, 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, 新西蘭白白化, 5-6 個月 | 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 |
| (包括人類, 獼猴, 兔) | |||||
| 兔 | 兔, 南美洲栗鼠, 5-8 月, 處女, 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, 新英格蘭, 白化變種 | 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, 胎兒 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 個月 | 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 KATP 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, 任一性別, 12-19 天, 3 個月, 和退休飼養者 | 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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