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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(xì)胞生物學(xué)研究的原代細(xì)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(duì)其組織消化和細(xì)胞收集的作用機(jī)制卻沒(méi)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(jī)選擇或基于過(guò)去的經(jīng)驗(yàn)，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(shí)驗(yàn)?zāi)康男拚桨敢垣@得更好的實(shí)驗(yàn)結(jié)果。
細(xì)胞分離程序的目的是zui大程度的獲得功能活性的單個(gè)細(xì)胞，而影響結(jié)果的原因有很多，包括（不限于）以下：
1、組織類(lèi)型
2、物種來(lái)源
3、動(dòng)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(shí)間
通常前四個(gè)原因沒(méi)辦法選擇，而剩下的原因一般由經(jīng)驗(yàn)決定以獲取zui適實(shí)驗(yàn)結(jié)果。
通過(guò)查閱文獻(xiàn)來(lái)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(shí)驗(yàn)數(shù)據(jù)搞得一頭霧水。這些巨大差異來(lái)源于組織胞外基質(zhì)的復(fù)雜而動(dòng)態(tài)的特性，以及過(guò)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(lèi)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(xì)胞收集知識(shí)和方法，描述了標(biāo)準(zhǔn)的實(shí)驗(yàn)步驟， 以便于制定一個(gè)*的細(xì)胞分離方案，同時(shí)列舉了一些相關(guān)的參考文獻(xiàn)。
下表列出多種組織的適宜消化酶（含參考文獻(xiàn)，以組織類(lèi)型和物種分類(lèi)，詳見(jiàn)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(dòng)物鱗片 | 皮膚 | 脾臟 |
干細(xì)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(xiàn)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(lèi)型的消化。
皮膚組織消化手冊(cè)
| 物種 | 詳細(xì)來(lái)源 | 細(xì)胞名稱(chēng) | 消化用酶 | 培養(yǎng)基 | 參考文獻(xiàn) |
| 犬 | 犬 | Epidermal | Collagenase: 0.35% | DMEM | Zheng, Y., Nace, A., Chen, W., Watkins, K., Sergott, L., Homan, Y., Vandeberg, J., Breen, M. and Stenn, K.: Mature Hair Follicles Generated from Dissociated Cells: A Universal Mechanism of Folliculoneogenesis., Dev Dyn 239, 2619-26, 2010 |
| Neutral Protease: 0.1% | |||||
| 青蛙 | 青蛙 | Epidermal | Trypsin: 0.18% | Barth's solution, CMF | Nishikawa, A., Shimizu-Nishikawa, K., and Miler, L.: Isolation, characterization, and in vitro culture of larval and adult epidermal cells of the frog Xenopus laevis, In Vitro Cell Dev Biol 26, 1128, 1990 |
| 山羊 | 山羊 | Dermis isolated aggrecan sensitive | Neutral Protease: 0.5% | DMEM | Deng, Y., Hu, J. and Athanasiou, K.: Isolation and Chondroinduction of a Dermis-Isolated, Aggrecan-Sensitive Subpopulation with High Chondrogenic Potential., Arthritis Rheum 56, 168, 2007 |
| Collagenase Type 2: 200 u/ml | |||||
| 人類(lèi) | 人類(lèi) | Skin | Neutral Protease: 5 u/ml | RPMI 1640 | Cheuk, S., Wiken, M., Blomqvist, L., Nylen, S., Talme, T., Stahle, M.and Eidsmo, L.:Epidermal Th22 and Tc17 Cells Form a Localized Disease Memory in Clinically Healed Psoriasis.,J Immunol 192, 3111, 2014 |
| Collagenase Type 3: 0.3% | |||||
| Deoxyribonuclease I: 0.005% | |||||
| 人類(lèi) | 人類(lèi) | Skin Fibroblasts | Collagenase Type 1: 1000 u/ml | DMEM | Douvaras, P., Wang, J., Zimmer, M., Hanchuk, S., O'Bara, M., Sadiq, S., Sim, F., Goldman, J. and Fossati, V.: Efficient Generation of Myelinating Oligodendrocytes from Primary Progressive Multiple Sclerosis Patients by Induced Pluripotent Stem Cells., Stem Cell Reports3, 250, 2014 |
| 人類(lèi) | 人類(lèi), 成年的 | Epidermal stem cells | Neutral Protease: 2 u/ml | DMEM | Nagel, S., Rohr, F., Weber, C., Kier, J., Siemers, F., Kruse, C., Danner, S., Brandenburger, M. and Matthiessen, A.: Multipotent Nestin-Positive Stem Cells Reside in the Stroma of Human Eccrine and Apocrine Sweat Glands and can be Propagated Robustly In Vitro., PLoS ONE 8, e78365, 2013 |
| Trypsin: 2.5% | |||||
| 人類(lèi) | 人類(lèi) | Fibroblasts | Collagenase animal free: 2% | HBSS | Karumbayaram, S., Lee, P., Azghadi, S., Cooper, A., Patterson, M., Kohn, D., Pyle, A., Clark, A., Byrne, J., Zack, J., Plath, K. and Lowry, W.: From Skin Biopsy to Neurons Through a Pluripotent Intermediate Under Good Manufacturing Practice Protocols, Stem Cells Transl Med 1, 36, 2012 |
| 人類(lèi) | 人類(lèi), 女性, 18-66 歲 | Fibroblasts | Trypsin: 0.2% | DMEM | Huschtscha, L., Napier, C., Noble, J., Bower, K., Au, A., Campbell, H., Braithwaite, A. and Reddel, R.: Enhanced Isolation of Fibroblasts From Human Skin Explants., Biotechniques 53, 239, 2012 |
| 人類(lèi) | 人類(lèi) | Epidermal | Collagenase: 0.35% | DMEM | Zheng, Y., Nace, A., Chen, W., Watkins, K., Sergott, L., Homan, Y., Vandeberg, J., Breen, M. and Stenn, K.: Mature Hair Follicles Generated from Dissociated Cells: A Universal Mechanism of Folliculoneogenesis., Dev Dyn 239, 2619-26, 2010 |
| Neutral Protease: 0.1% | |||||
| 人類(lèi) | 人類(lèi) | skin and keloid progenitor cells | Collagenase Type 1: 0.4% | PBS | Zhang, Q., Yamaza, T., Kelly, AP, Shi, S., Wang, S., Brown, J., Wang, L., French, S., Shi, S. and Le, A.: Tumor-like Stem Cells Derived from Human Keloid are Governed by the Inflammatory Niche Driven by IL-17/IL-6 Axis., PLoS ONE 4, e7798, 2009 |
| Neutral Protease: 0.3% | |||||
| 人類(lèi) | 人類(lèi) | Fibroblasts | Collagenase Type 1: 100-320 u/ml | DMEM | Tuan, T., Hwu, P., Ho, W., Yiu, P., Chang, R., Wysocki, A. and Benya, P.: Adenoviral Overexpression and Small Interfering RNA Suppression Demonstrate that Plasminogen Activator Inhibitor-1 Produces Elevated Collagen Accumulation in Normal and Keloid Fibroblasts., Am J Pathol 173, 1311-25, 2008 |
| 人類(lèi) | 人類(lèi), 6-12 歲 | Dermal fibroblasts | Neutral Protease: 0.1% | DMEM | Chen, F., Zhang, W., Bi, D., Liu, W., Wei, X., Chen, F., Zhu, L., Cui, L. and Cao, Y.: Clonal Analysis of Nestin(-) Vimentin(+) Multipotent Fibroblasts Isolated from Human Dermis., J Cell Sci120, 2875, 2007 |
| Collagenase Type 1: 0.1% | |||||
| 人類(lèi) | 人類(lèi) | Hair follicular epithelial | Neutral Protease: 1.25% | DMEM/F-12 | Yu Hong, Fang Dong, Kumar Suresh M, Li Ling, Nguyen Thiennga K, Acs Geza, Herlyn Meenhard, Xu Xiaowei: Isolation of a novel population of multipotent adult stem cells from human hair follicles, Am J Pathol 168, 1879-88, 2006 |
| Trypsin: 0.25% | |||||
| 人類(lèi) | 人類(lèi) | fibroblasts | Collagenase: 0.5% | DMEM/F12 | Clark, R., Chong, B., Mirchandani, N., Yamanaka, K., Murphy, G., Dowgiert, R. and Kupper, T.: A Novel Method for the Isolation of Skin Resident T cells from Normal and Diseased Human Skin., J Invest Dermatol 126, 1059, 2006 |
| Trypsin: 0.25% | |||||
| 人類(lèi) | 人類(lèi), 新生的 | Keratinocytes | Neutral Protease: 0.4% | DMEM | Li, A., Pouliot, N., Redvers, R., and Kaur, P.: Extensive tissue-regenerative capacity of neonatal human keratinocytes stem cells and their progeny, J Clin Invest 113, 390-400, 2004 |
| Collagenase: 0.3% | |||||
| 人類(lèi) | 人類(lèi) | Mast cells | Neutral Protease: 0.05% | RPMI | Babina Magda, Guhl Sven, Starke Andrae, Kirchhof Loreen, Zuberbier Torsten, Henz Beate M: Comparative cytokine profile of human skin mast cells from two compartments--strong resemblance with monocytes at baseline but induction of IL-5 by IL-4 priming, J Leukoc Biol 75, 244-52, 2004 |
| Collagenase Type 4: 1.0% | |||||
| 人類(lèi) | 人類(lèi), 成年的 | Dermal fibroblasts | Collagenase: 0.5-1.0% | DMEM | Wang, H., Van Blitterswijk, C., Bertrand-De Haas, M., Schuurman, A. and Lamme, E.:Improved Enzymatic Isolation of Fibroblasts for the Creation of Autologous Skin Substitutes., In Vitro Cell Dev Biol Anim 40, 268, 2004 |
| 人類(lèi) | 人類(lèi) | Fibroblasts | Trypsin: 0.25% | DMEM | Tuan Tai-Lan, Wu Huayang, Huang Eunice Y, Chong Sheree S N, Laug Walter, Messadi Diana, Kelly Paul, Le Anh: Increased plasminogen activator inhibitor-1 in keloid fibroblasts may account for their elevated collagen accumulation in fibrin gel cultures, Am J Pathol 162, 1579-89, 2003 |
| 人類(lèi) | 人類(lèi) | Keratinocytes, fibroblasts, endothelial | Neutral Protease: see reference | see reference | Supp Dorothy M, Wilson-Landy Kaila, Boyce Steven T: Human dermal microvascular endothelial cells form vascular analogs in cultured skin substitutes after grafting to athymic mice, FASEB J 16, 797-804, 2002 |
| Collagenase: see reference | |||||
| 人類(lèi) | 人類(lèi), 成年的 | Keratinocytes | Neutral Protease: 0.25% | PBS | Baudoux, B., Castanares-Zapatero, D., Leclercq-Smekens, M., Berna, N., and Poumay, Y.: The Tetraspanin CD9 Associates with the Integrin a6B4 in Cultured Human Epidermal Keratinocytes and is Involved in Cell Motility, Eur J Cell Biol 79, 41, 2000 |
| 人類(lèi) | 人類(lèi) | Keratinocytes | Neutral Protease: 0.25% | see reference | Hybbinette S, Bostrom M, Lindberg K: Enzymatic dissociation of keratinocytes from human skin biopsies for in vitro cell propagation, Exp Dermatol 8, 30-8, 1999 |
| Trypsin: 0.17% | |||||
| Thermolysin: 0.05% | |||||
| 人類(lèi) | 人類(lèi) | Human skin mast cells | Neutral Protease: 0.1% | PBS | Grutzkau A, Kruger-Krasagakes S, Baumeister H, Schwarz C, Kogel H, Welker P, Lippert U, Henz BM, Moller A: Synthesis, storage, and release of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) by human mast cells: implications for the biological significance of VEGF206, Mol Biol Cell 9, 875-84, 1998 |
| Collagenase Type 1: see reference | |||||
| 人類(lèi) | 人類(lèi) | Epidermal | Collagenase Type 2: 0.1% | PBS | Reece J C, Handley A J, Anstee E J, Morrison W A, Crowe S M, Cameron P U: HIV-1 selection by epidermal dendritic cells during transmission across human skin, J Exp Med 187, 1623-31, 1998 |
| Neutral Protease: 0.5-1.0% | |||||
| 人類(lèi) | 人類(lèi) | Keratinocytes | Trypsin: 0.05% | Dulbecco's PBS | Judd, D., Battista, P., and Behm, D.: Culture of Human Keratinocytes in Defined Serum-Free Medium, Focus 19 (1), 2, 1997 |
| 人類(lèi) | 人類(lèi) | Epidermis plus dermis | Trypsin: 0.125% | DMEM, M199 | Harley, C., and Sherwood, S.: Methods in Molecular Biology, Basic Cell Culture Protocols, 2nd ed. Vol. 75,Pollard, J., and Walker, J., Humana Press, 23, 1997 |
| Abdomen or inner forearm | |||||
| 人類(lèi) | 人類(lèi) | Keratinocytes | Trypsin: 0.25% | DMEM | Regnier, M: Culture of Human Karatinocytes,Cell & Tissue Culture: Laboratory Procedures Vol. 1,Doyle, A., Griffiths, J., and Newell, D., John Wiley and Sons Ltd, 11B:4.1, 1995 |
| 人類(lèi) | 人類(lèi), 成年的, 女性, 乳房皮膚 | Keratinocytes | Trypsin: 0.25% | PBS | Hirel, B., Chesne, C., Pailgheret, J., and Guillouzo, A.: Expression of Differentiation Markers in Human Adult Keratinocytes Cultured in Submerged Conditions, In Vitro Cell Dev Biol 30A, 372, 1994 |
| 人類(lèi) | 人類(lèi), 新生兒包皮 | Fibroblasts | Trypsin: 0.25% | DMEM | Hansbrough, J., Cooper, M., Cohen, R., Spielvogel, R., Greenleaf, G., Bar, R., and Naughton, G.: Evaluation of a Biodegradable Matrix Containing Cultured Human Fibroblasts as a Dermal Replacement Beneath Meshed Skin Grafts on Athymic Mice, Surgery 111 (1), 438, 1992 |
| 人類(lèi) | 人類(lèi), 18-50 歲 | Sweat duct | Collagenase: 0.03% | MEM | Bell, C. and Quinton, P.: Effects of Media Buffer Systems on Growth and Electrophysiologic Characteristics of Cultured Sweat Duct Cells, In Vitro Cell Dev Biol 27, 47, 1991 |
| 人類(lèi) | 人類(lèi) | Fibroblasts | Trypsin: 0.25% | CMF solution | Limat, A., Hunziker, T., Boillat, C., Noser, F., and Wiesmann, U.: Postmitotic Human Dermal Fibroblasts Preserve Intact Feeder Properties for Epithelial Cell Growth After Long-Term Cryopreservation, In Vitro Cell Dev Biol 26, 709, 1990 |
| 人類(lèi) | 人類(lèi) | Sweat gland | Collagenase Type 2: 0.015% | (see reference) | Krouse, M., Hagiqara, G., Chen, J., Lewiston, N., and Wine, J.: Ion Channels in Normal Human and Cystic Fibrosis Sweat Gland Cells, Am J Physiol 257, C129, 1989 |
| 人類(lèi) | 人類(lèi), 年齡16-30 | Human sweat duct | Collagenase Type 2: 0.2% | RPMI 1640 | Pedersen, P.: Human Sweat Duct Cells in Primary Culture. Basic Bioelectric Properties of Cultures Derived From Normals and Patients with Cystic Fibrosis, In Vitro Cell Dev Biol 25 (4), 342, 1989 |
| (see reference) | |||||
| 人類(lèi) | 人類(lèi) | Melanocytes, skin/ foreskin | Trypsin: 0.25% | PBS | Peacocke, M., Yaar, M., Mansur, C., Chao, M., and Gilchrest, B.: Induction of Nerve Growth Factor Receptors on Cultured Human Melanocytes, Proc Natl Acad Sci U S A 85, 5282, 1988 |
| 人類(lèi) | 人類(lèi) | Human sweat glands | Collagenase: 0.2% | HBSS | Lee, C., Carpenter, F., Coaker, T., and Kealey, T.: The Primary Culture of Epithelia From the Secretory Coil and Collecting Duct of Normal Human and Cystic Fibrotic Eccrine Sweat Glands, J Cell Sci 83, 103, 1986 |
| 人類(lèi) | 人類(lèi) | Keratinocytes | Trypsin: 0.25% | DMEM | Dover, R. and Potten, C.: Cell Cycle Kinetics of Cultured Human Epidermal Keratinocytes, J Invest Dermatol 80, 423, 1983 |
| 人類(lèi) | 人類(lèi), 成年的 | Epidermal keratinocytes | Trypsin: 0.25% | Eagle's MEM | Alitalo K, Kuismanen E, Myllyla R, Kiistala U, Asko-Seljavaara S, Vaheri A: Extracellular matrix proteins of human epidermal keratinocytes and feeder 3T3 cells, J Cell Biol 94, 497-505, 1982 |
| Collagenase: 0.2% | |||||
| Deoxyribonuclease I: 0.001% | |||||
| 人類(lèi) | 人類(lèi), fetal | 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 |
| 人類(lèi) | 人類(lèi), 18-30歲, 男性, 女性 | Fibroblasts | Trypsin: 0.1% | HBSS | McCoy, B., Galdun, J., and Cohen, I.: Effects of Density and Cellular Aging On Collagen Synthesis and Growth Kinetics in Keloid and Normal Skin Fibroblasts, In Vitro 18 (1), 79, 1982 |
| 人類(lèi) | 人類(lèi), 新生兒 | Keratinocytes | Trypsin: 0.3% | DMEM | Liu, S., Eaton, M., and Karasek, M.: Growth Characteristics of Human Epidermal Keratinocytes from Newborn Foreskin in Primary and Serial Cultures, In Vitro 15 (10), 813, 1979 |
| 人類(lèi) | 人類(lèi) | Keratinocytes | Trypsin: 0.25% | (see reference) | Rheinwald, J., and Green, H.: Serial C*tion of Strains of Human Epidermal Kerotinocytes: The Formation of Keratinizing Colonies from Single Cells, Cell 6, 331, 1975 |
| 小鼠 | 小鼠 | Epidermal | Neutral Protease: 0.5% | DMEM | Gu, D., Fan, Q and Xie, J.: Cell Population Analyses During Skin Carcinogenesis., J Vis Exp , e50311, 2013 |
| Collagenase Type 4: 0.1% | |||||
| 小鼠 | 小鼠 | Epithelial Stem | Trypsin: 0.25% | HBSS | Jensen, U., Ghazizadeh, S. and Owens, D.: Isolation and Characterization of Cutaneous Epithelial Stem Cells., Methods Mol Biol 989, 61-9, 2013 |
| Collagenase Type 1: 0.4% | |||||
| 小鼠 | 小鼠 | Dermal fibroblasts | Collagenase: 400 u/ml | AminoMAX II | Scheibye-Knudsen, M., Ramamoorthy, M., Sykora, P., Maynard, S., Lin, P., Minor, R., Wilson, D., Cooper, M., Spencer, R., de Cabo, R., Croteau, D. and Bohr, V.: Cockayne Syndrome Group B Protein Prevents the Accumulation of Damaged Mitochondria by Promoting Mitochondrial Autophagy., J Exp Med 209, 855, 2012 |
| 小鼠 | 小鼠 | Epidermal and dermal | Collagenase Type 4: 0.18% | PBS | King, I., Kroenke, M. and Segal, B.: GM-CSF-Dependent, CD103+ Dermal Dendritic Cells Play a Critical Role in Th Effector Cell Differentiation After Subcutaneous Immunization., J Exp Med 207, 953, 2010 |
| Collagenase/Dispase: 0.18% | |||||
| 小鼠 | 小鼠, 2-3 天 | Dermal fibroblasts | Collagenase Type 2: 0.04% | DME | Farina, G., York, M., Di Marzio, M., Collins, C., Meller, S., Homey, B., Rifkin, I., Marshak-Rothstein, A., Radstake, T. and Lafyatis, R.: Poly(I:C) Drives Type I IFN- and TGF尾-Mediated Inflammation and Dermal Fibrosis Simulating Altered Gene Expression in Systemic Sclerosis., J Invest Dermatol 130, 2583-93, 2010 |
| Trypsin: 0.025% | |||||
| 小鼠 | 小鼠 | Dermal | Collagenase Type 3: 0.3% | HBSS | Eidsmo, L., Allan, R., Caminschi, I., Van Rooijen, N., Heath, W. and Carbone, F.:Differential Migration of Epidermal and Dermal Dendritic Cells During Skin Infection., J Immunol 182, 3165, 2009 |
| Deoxyribonuclease I: 0.0005% | |||||
| 小鼠 | 小鼠, 新生的 | Dermal | Collagenase: 0.35% | PBS | Crigler Lauren, Kazhanie Amita, Yoon Tae-Jin, Zakhari Julia, Anders Joanna, Taylor Barbara, Virador VictoriaM: Isolation of a mesenchymal cell population from murine dermis that contains progenitors of multiple cell lineages, FASEB J 21, 2050-63, 2007 |
| Deoxyribonuclease I: see reference | |||||
| 小鼠 | 小鼠 | Dermal fibroblasts | Trypsin: 0.25% | DMEM | Baxter Ruth M, Crowell Thomas P, McCrann Margaret E, Frew Erica M, Gardner Humphrey: Analysis of the tight skin (Tsk1/+) mouse as a model for testing antifibrotic agents,Lab Invest 85, 1199-209, 2005 |
| Collagenase Type 1: 0.25% | |||||
| 小鼠 | 小鼠, 新生的 | Microvascular endothelial | Neutral Protease: 0.005% | DMEM | Cha, S., Talavera, D., Demir, E., Nath, A. and Sierra-Honigmann, M.: A Method of Isolation and Culture of Microvascular Endothelial Cells from Mouse Skin., Microvasc Res 70, 198, 2005 |
| Collagenase Type 1: 4% | |||||
| 小鼠 | 小鼠, male 6-24 week | Skin side population | Collagenase Type 4: 0.2% | PBS | Montanaro F, Liadaki K, Volinski J, Flint A, and Kunkel LM.: Skeletal muscle engraftment potential of adult mouse skin side population cells, Proc Natl Acad Sci U S A 100, 9336, 2003 |
| Neutral Protease: 1.2 u/ml | |||||
| 小鼠 | 小鼠 | Ear epidermal | Trypsin: 0.1% | PBS | Takanami-Ohnishi Yoko, Amano Shinya, Kimura Sadao, Asada Sachie, Utani Atsushi, Maruyama Masumi, Osada Hiroyuki, Tsunoda Hajime, Irukayama-Tomobe Yoko, Goto Katsutoshi, Karin Michael, Sudo Tatsuhiko, Kasuya Yoshitoshi: Essential role of p38 mitogen-activated protein kinase in contact hypersensitivity, J Biol Chem 277, 37896-903, 2002 |
| Collagenase: 0.2% | |||||
| 小鼠 | 小鼠, 3-7 月 | Fibroblasts, mesangial, smooth muscle | Trypsin: 0.25% | DMEM | Bradshaw AD, Francki A, Motamed K, Howe C, Sage EH: Primary mesenchymal cells isolated from SPARC-null mice exhibit altered morphology and rates of proliferation, Mol Biol Cell 10, 1569-79, 1999 |
| Collagenase: see reference | |||||
| Soybean Trypsin Inhibitor: .05% | |||||
| 豬 | 豬, 3-4 月 | Synovial membrane and skin stem cells | Collagenase: 0.2% | DMEM | Ando, W., Kutcher, J., Krawetz, R., Sen, A., Nakamura, N., Frank, C. and Hart, D.: Clonal Analysis of Synovial Fluid Stem Cells to Characterize and Identify Stable Mesenchymal Stromal Cell/Mesenchymal Progenitor Cell Phenotypes in a Porcine Model: A Cell Source with Enhanced Commitment to the Chondrogenic Lineage., Cytotherapy 16, 776, 2014 |
| 豬 | 豬, 2-6 月 | Keratinocytes | Neutral Protease: 0.25% | Dulbecco-Vogt MEM | Regauer S, Compton C: Cultured porcine epithelial grafts: an improved method, J Invest Dermatol 94, 230-4, 1990 |
| 大鼠 | Wistar大鼠, 4-8 天 | Dermal fibroblasts and keratinocytes | Trypsin: 0.25% | Ham's F-12 | Sugihara, H and Toda, S: Primary Tissue Intact and Dissociated Cell Culture,Cell & Tissue Culture: Laboratory Procedures Vol. 1,Doyle, A., Griffiths, J., and Newell, D., John Wiley and Sons, Ltd., 3A:2.1, 1995 |
| 大鼠 | SD大鼠, 雄性, 40-60 天 | Sebaceous | Trypsin: 0.2% | DMEM with FBS | Laurent, S., Mednieks, M., and Rosenfield, R.: Growth of Sebaceous Cells in Monolayer Culture, In Vitro Cell Dev Biol 28, 83, 1992 |
| PBS | |||||
| 大鼠 | 大鼠, 新生的, 產(chǎn)后的12-24 小時(shí) | Fibroblasts | Trypsin: 0.2% | HEPES buffered DMEM | Acheson, A., Barker, P., Alderson, R., Miller, F., and Murphy, R.: Detection of Brain-Derived Neurotrophic Factor-like Activity in Fibroblasts and Schwann Cells: Inhibition by Antibodies to NGF, Neuron 7, 265, 1991 |
| 大鼠 | 大鼠, 白化變種, 一天大, CFN | Keratinocytes | Trypsin: 1% | EBSS | Vaughan, F., Gray, R., and Bernstein, I.: Growth and Differentiation of Primary Rat Keratinocytes on Synthetic Membranes, In Vitro Cell Dev Biol 22 (3), 141, 1986 |
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