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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。
生殖组织消化手册
| 物种 | 详细来源 | 细胞名称 | 消化用酶 | 培养基 | 参考文献 |
| 牛 | 牛 | Corpus leuteal cells | Collagenase Type 4: 420 u/ml | M-199 | Levy N, Gordin M, Mamluk R, Yanagisawa M, Smith M F, Hampton J H, Meidan R:Distinct cellular localization and regulation of endothelin-1 and endothelin-converting enzyme-1 expression in the bovine corpus luteum: implications for luteolysis, Endocrinology 142, 5254-60, 2001 |
| 牛 | 牛, 雌性 | Leuteal | Collagenase Type 1: 0.2% | Ham's F-12 | Tsang PC, Poff JP, Boulton EP, Condon WA: Four-day-old bovine corpus luteum: progesterone production and identification of matrix metalloproteinase activity in vitro, Biol Reprod 53, 1160-8, 1995 |
| 牛 | 牛犊,中晚期妊娠 | Fibroblasts | Collagenase: 0.1% | Medium 199 | Coplen, D., Howard, P., Duckett, J., Snyder, H., and Macarak, E.:Characterization of a Fibroblast Cell From the Urinary Bladder Wall, In Vitro Cell Dev Biol 30A, 604, 1994 |
| 牛 | 牛 | Epithelial | Collagenase Type 2: 0.1% | DMEM/EBSS | Munson, L., Chandler, S., and Schlafer, D.: Long-Term Culture of Bovine Trophoblastic Cells, J Tiss Cul Meth 11 (3), 123, 1988 |
| Endometrial | |||||
| 牛 | 牛 | Interna & corpus luteum | Pronase: 0.1% | Moscona's BSS | Marcus, G., Connor, L., Domingo, M., Tsang, B., Downey, B., and Ainsworth, L.:Enzymatic Dissociation of Ovarian and Uterine Tissues, Endocr Res 10, 151, 1984 |
| (包括猪，人类) | Endometrium | ||||
| Ovarian | |||||
| Uterine | |||||
| 犬 | 犬 | Amniotic membrane MSC | Trypsin: 0.25% | LG-DMEM | Park, S., Seo, M., Kim, H. and Kang, K.: Isolation and Characterization of Canine Amniotic Membrane-Derived Multipotent Stem Cells., PLoS ONE 7, e44693, 2012 |
| Collagenase Type 1: 0.2% | |||||
| 鸡 | 鸡, 家鸡,20-30 周 | Primary follicles | Trypsin: 0.15% | Dulbecco's phosphate buffered saline | Du Meihong, Han Haitang, Jiang Bin, Zhao Chen, Qian Changsong, Shen Haiyan, Xu Yan, Li Zandong: An efficient isolation method for domestic hen (Gallus domesticus) ovarian primary follicles, J Reprod Dev 52, 569-76, 2006 |
| Collagenase Type 1: 0.125% | |||||
| 青蛙 | 非洲蟾蜍 光滑的 | Oocytes | Collagenase: 0.2% | Barth's solution | Chatzidaki, A., D'Oyley, J., Gill-Thind, J., Sheppard, T. and Millar, N.: The Influence of Allosteric Modulators and Transmembrane Mutations on Desensitisation and Activation of alpha7 Nicotinic Acetylcholine Receptors., Neuropharmacol 97, 75-85, 2015 |
| 青蛙 | 非洲蟾蜍 光滑的, 雌性 | Oocytes | Collagenase: 0.2% | see reference | Mruk, K. and Kobertz, W.: Discovery of a Novel Activator of KCNQ1-KCNE1 K Channel Complexes., PLoS ONE 4, e4236, 2009 |
| 青蛙 | 非洲蟾蜍 光滑的, 雌性 | Oocytes | Collagenase: 0.5% | Barth's solution, CF | Cohen, S., Au, S. and Pante, N.: Microinjection of Xenopus Laevis Oocytes., J Vis Exp 24, 1106, 2009 |
| 青蛙 | 非洲蟾蜍 光滑的, 雌性 | Oocytes | Collagenase Type 1: 0.2% | see reference | Pannaccione Anna, Castaldo Pasqualina, Ficker Eckhard, Annunziato Lucio, Taglialaa Maurizio: Histidines 578 and 587 in the S5-S6 linker of the human Ether-a-gogo Related Gene-1 K+ channels confer sensitivity to reactive oxygen species, J Biol Chem 277, 8912-9, 2002 |
| 青蛙 | 非洲蟾蜍 光滑的, 雌性 | Oocytes | Collagenase Type 1: 0.2% | CF Medium | Alagem, N., Dvir, M., and Reuveny, E.: Mechanism of Ba2+ Block of a Mouse Inwardly Rectifying K+ Channel: Differential Contribution by Two Discrete Residues, J Physiol 534 (2), 381, 2001 |
| 青蛙 | 非洲蟾蜍 | Oocytes | Collagenase Type 1: 1% | (see reference) | Tian, J., Kim, S., Heilig, E., and Ruderman, J.: Identification of XPR-1, A Progesterone Receptor Required for Xenopus Oocyte Activation, Proc Natl Acad Sci U S A 97, 14358, 2000 |
| 青蛙 | 非洲蟾蜍 光滑的, 雌性 | Oocytes | Collagenase: 0.1% | Barth's solution, CF | Karkanias, N, and Papke, R.: Subtype-Specific Effects of Lithium on Glutamate Receptor Function, J Neurophysiol 81, 1506, 1999 |
| 青蛙 | 非洲蟾蜍 光滑的, 成熟的雌性 | Oocytes | Collagenase: 0.2% | CF Medium | Moriarty, T., Gillo, B., Carty, D., Premont, R., Landau, E., Iyengar, R.: Beta gamma Subunits of GTP-Binding Proteins Inhibit Muscarinic Receptor Stimulation of Phospholipase C, Proc Natl Acad Sci U S A 85, 8865, 1988 |
| 马 | 马，中国的 | Ovary | Trypsin: 5% | Dialyzed fetal calf serum, 10% and 0.5M Methotrexate | Wallis, R., and Drickamer, K.: Molecular Determinants of Oligomer Formation and Complement Fixation in Mannose-Binding Proteins, J Biol Chem 274 (6), 3580, 1999 |
| 人类 | 人类 | Endothelial colony forming | Collagenase Type 1: 0.1% | HBSS | Pa, J., Seppanen, E., Chong, M., Yeo, J., Teo, E., Chan, J., Fisk, N. and Khosrotehrani, K.: Prospective Surface Marker-Based Isolation and Expansion of Fetal Endothelial Colony-Forming Cells from Human Term Placenta., Stem Cells Transl Med 2, 839-47, 2013 |
| Deoxyribonuclease I: 0.1% | |||||
| Neutral Protease: 0.075% | |||||
| 人类 | 人类 | Uterine epithelial | Pancreatin: 0.34% | HBSS | Pa, M., Ghosh, M., Fahey, J. and Wira, C.: Uterine Epithelial Cells Specifically Induce Interferon-Stimulated Genes in Response to Polyinosinic-Polycytidylic Acid Independently of Estradiol., PLoS ONE 7, e35654, 2012 |
| Hyaluronidase: 0.01% | |||||
| Collagenase: 0.16% | |||||
| 人类 | 人类 | Mesenchymal stem | Collagenase Type 1: 0.4% | DMEM/F12 | Shalini, V., Pratheep, S., Muhammad, A., Sharmili, V., Elizabeth, G. and Rajesh, R.: Generation and Characterisation of Human Mesenchymal Stem Cells Derived From Umbilical Cord and Placenta, Regenerative Research 1, 48, 2012 |
| Deoxyribonuclease I: 0.01% | |||||
| 人类 | 人类 | Mesenchymal stem | Collagenase Type 2: 10% | CMF-DPBS | Steigman, S. and Fauza, D.: Isolation of Mesenchymal Stem Cells from Amniotic Fluid and Placenta., Curr Protoc Stem Cell Biol Chapter 1, Unit 1E.2, 2007 |
| Neutral Protease: see reference | |||||
| 人类 | 人类, 女性 | Decidual | Collagenase: 0.25% | DMEM/F12 | Lockwood, C., Arcuri, F., Toti, P., Felice, C., Krikun, G., Guller, S., Buchwalder, L. and Schatz, F.: Tumor Necrosis Factor-Alpha and Interleukin-1 Beta Regulate Interleukin-8 Expression in Third Trimester Decidual Cells: Implications for the Genesis of Chorioamnionitis., Am J Pathol 169, 1294-302, 2006 |
| Deoxyribonuclease I: 6.25 u/ml | |||||
| 人类 | 人类 | Extravillanous Trophoblasts and Decidual Stromal | Trypsin: 0.1-0.25% | HBSS RPMI | Spessotto, P., Bulla, R., Danussi, C., Radillo, O., Cervi, M., Monami, G., Bossi, F., Tedesco, F., Doliana, R. and Colombatti, A.: EMILIN1 Represents a Major Stromal Element Determining Human Trophoblast Invasion of the Uterine Wall., J Cell Sci119, 4574, 2006 |
| Collagenase Type 1: 0.1% | |||||
| Deoxyribonuclease I: 0.02-0.05% | |||||
| 人类 | 人类 | Sertoli cells | Trypsin: 2.5% | DMEM/F-12 | Teng Yan, Xue Wu-jun, Ding Xiao-ming, Feng Xin-shun, Xiang He-li, Jiang Ya-zhuo, Tian Pu-xun: Isolation and culture of adult Sertoli cells and their effects on the function of co-cultured allogeneic islets in vitro, Chin Med J (Engl) 118, 1857-62, 2005 |
| Collagenase Type 1: 2% | |||||
| Hyaluronidase: 1% | |||||
| 人类 | 人类, 女性 | Uterine epithelial | Pancreatin: 0.34% | HBSS | Meter, R., Wira, C. and Fahey, J.: Secretion of Monocyte Chemotactic Protein-1 by Human Uterine Epithelium Directs Monocyte Migration in Culture., Fertil Steril 84, 191, 2005 |
| Hyaluronidase: 0.01% | |||||
| Collagenase: 0.16% | |||||
| 人类 | 人类, 女性, 34-51 岁 | Endometrial epithelial and stromal cells | Collagenase Type 3: 0.03% | DMEM/F-12 | Chan Rachel W S, Schwab Kjiana E, Gargett Caroline E: Clonogenicity of human endometrial epithelial and stromal cells, Biol Reprod 70, 1738-50, 2004 |
| Deoxyribonuclease I: 0.004% | |||||
| 人类 | 人类 | Endothelial placental | Collagenase Type 1: 0.2% | DMEM | Wang Xin, Athayde Neil, Trudinger Brian: Microvascular endothelial cell activation is present in the umbilical placental microcirculation in fetal placental vascular disease, Am J Obstet Gynecol 190, 596-601, 2004 |
| Trypsin: 0.2% | |||||
| Deoxyribonuclease I: 0.1% | |||||
| 人类 | 人类 | Amnion epithelial and fibroblast | Trypsin: 0.125% | PBS | Sun Kang, Myatt Leslie: Enhancement of glucocorticoid-induced 11beta-hydroxysteroid dehydrogenase type 1 expression by proinflammatory cytokines in cultured human amnion fibroblasts, Endocrinology 144, 5568-77, 2003 |
| Collagenase: 0.1% | |||||
| Deoxyribonuclease I: 0.02% | |||||
| 人类 | 人类 | Uterine epithelial cells | Pancreatin: 0.34% | HBSS | Fahey John V, Wira Charles R: Effect of menstrual status on antibacterial activity and secretory leukocyte protease inhibitor production by human uterine epithelial cells in culture, J Infect Dis 185, 1606-13, 2002 |
| Collagenase: 0.16% | |||||
| Hyaluronidase: 0.16% | |||||
| 人类 | 人类,胚胎 | Endothelial | Collagenase Type 1/2/4: 0.1% | DMEM | Oberlin, E., Tavian, M., Blazsek, I., and Peault, B.: Blood-forming potential of vascular endothelium in the human embryo, Development 129, 4147, 2002 |
| Hematopoietic | |||||
| Stromal | |||||
| 人类 | 人类, 女性 | Mesothelial | Collagenase Type 1: 0.1% | Eagle's MEM | Witz Craig A, Allsup Karen T, Montoya-Rodriguez Iris A, Vaughn Shelby L, Centonze Victoria E, Schenken Robert S: Culture of menstrual endometrium with peritoneal explants and mesothelial monolayers confirms attachment to intact mesothelial cells, Hum Reprod 17, 2832-8, 2002 |
| Deoxyribonuclease I: 0.05% | |||||
| 人类 | 人类 | Stem, embryonic | Neutral Protease: 0.01% - 0.02% | DMEM | Zhang, S., Wernig, M., Duncan, I., Brustle, O., and Thomson, J.: In VitroDifferentiation of Transplantable Neural Precursors from Human Embryonic Stem Cells, Nat Biotechnol 19, 1129, 2001 |
| 人类 | 人类, 20-40 岁 | Endometrium epithelial and stromal cells | Collagenase Type 1: 0.2% | HBSS | Arnold, J., Kaufman, D., Seppala, M., and Lessey, B.: Endometrial stromal cells regulate epithelial cell grownth in vitro: a new co-culture model, Hum Reprod 16, 836, 2001 |
| 人类 | 人类 | Chorionic villi | Trypsin: see reference | HBSS | Yusuf RZ, Naeem R: Cytogenetic studies of spontaneous miscarriages: a seven year study to compare significance of primary vs. secondary culture methods for assessment of fetal karyotype yield and maternal cell contamination, Early Pregnancy 5, 121-31, 2001 |
| Collagenase Type 3: 100 u/ml | |||||
| 人类 | 人类 | Endometrial endothelial cells | Collagenase Type 1: 0.2% | McCoys medium | Nikitenko LL, MacKenzie IZ,Rees MC, Bicknell R: Adrenomedullin is an autocrine regulator of endothelial growth in human endometrium, Mol Hum Reprod 6, 811, 2000 |
| 人类 | 人类, 女性, 20-40 岁 | Microvascular endothelial cells | Collagenase Type 2: 0.2% | PBS | Gargett CE, Bucak K, Rogers PA: Isolation, characterization and long-term culture of human myometrial microvascular endothelial cells, Hum Reprod 15, 293-301, 2000 |
| Deoxyribonuclease I: 0.0015% | |||||
| Trypsin: 0.05% | |||||
| 人类 | 人类,女性 | Endometrial epithelial cells | Collagenase Type 3: 45 u/ml | DMEM/F-12 | Zhang J, Lathbury LJ, Salamonsen LA: Expression of the chemokine eotaxin and its receptor, CCR3, in human endometrium, Biol Reprod 62, 404-11, 2000 |
| Deoxyribonuclease I: .00035% | |||||
| 人类 | 人类 | Theca cells | Collagenase Type 1: 0.3% | PBS | Runesson E, Ivarsson K, Janson P, Brannstrom M: Gonadotropin- and cytokine-regulated expression of the chemokine interleukin 8 in the human preovulatory follicle of the menstrual cycle, J Clin Endocrinol Metab 85, 4387-95, 2000 |
| Deoxyribonuclease I: 0.0005% | |||||
| Hyaluronidase: 0.1% | |||||
| 人类 | 人类 | Corpus luteum cells | Collagenase Type 2: 0.25% | PBS | Friden BE, Runesson E, Hahlin M, Brannstrom M: Evidence for nitric oxide acting as a luteolytic factor in the human corpus luteum, Mol Hum Reprod 6, 397-403, 2000 |
| Deoxyribonuclease I: .005% | |||||
| 人类 | 人类, 女性, 25-45 岁 | Luteal cells | Collagenase Type 2: 0.25% | PBS | Friden BE, Hagstrom H, Lindblom B, Sjoblom P, Wallin A, Brannstrom M, Hahlin M: Cell characteristics and function of two enriched fraction of human luteal cells prolonged culture, Mol Hum Reprod 5, 714-9, 1999 |
| Deoxyribonuclease I: 0.005% | |||||
| 人类 | 人类, 女性, 25-41 岁 | Follicles | Collagenase Type 2: 0.025-0.1% | EBSS | Hovatta O, Wright C, Krausz T, Hardy K, Winston RM: Human primordial, primary and secondary ovarian follicles in long-term culture: effect of partial isolation, Hum Reprod 14, 2519-24, 1999 |
| 人类 | 人类 | Placental | Deoxyribonuclease I: 0.04% | PSS | Bradbury., R., Sunn, K., Crossley, M., Bai, M., Brown, E., Delbridge, D., and Conigrave, A.: Expression of the Parathyroid Ca 2+ - Sensing Receptor in Cytotrophoblasts From Human Term Placenta, J Endocrinol 156, 425-430, 1998 |
| Collagenase Type 2: 0.1% | |||||
| 人类 | 人类, 女性 | Endometrial stromal cells | Collagenase: 4000 u/ml | DMEM/F-12 | Huang JC, Liu DY, Dawood MY: The expression of vascular endothelial growth factor isoforms in cultured human endometrial stromal cells and its regulation by 17beta-oestradiol, Mol Hum Reprod 4, 603-7, 1998 |
| 人类 | 人类 | Stromal endometrial | Collagenase Type 3: 45 u/ml | DMEM/Ham's F12 | Zhang J, Nie G, Jian W, Woolley DE, Salamonsen LA: Mast cell regulation of human endometrial matrix metalloproteinases: A mechanism underlying menstruation, Biol Reprod 59, 693-703, 1998 |
| Deoxyribonuclease I: .00035% | |||||
| 人类 | 人类 | Epithelial, fallopian tube | Collagenase Type 1: 1% | Medium 199 | Takeuchi, K., Maruyama, I., Yamamoto, S., Oki, T., Nagata, Y.: Isolation and Monolayer Culture of Human Fallopian Tube Epithelial Cells, In Vitro Cell Dev Biol 27, 720, 1991 |
| 人类 | 人类 | Trophoblasts, placental | Trypsin: 0.25% | EBSS, CMF | Branchaud, C.L., Goodyer, C.G., Guyda, H.J. and Lefebvre, Y.: A Serum-Free System for Culturing Human Placental Trophoblasts, In Vitro Cell Dev Biol 26, 865, 1990 |
| 人类 | 人类 | Trophoblasts, placental | Trypsin: 0.25% | PBS | Jie, Z., Fey, S., Hager, H., Hollsberg, P., Ebbesen, P., and Larsen, P.: Markers For Human Placental Trophoblasts in Two-Dimensional Gel Electrophoresis, In Vitro Cell Dev Biol 26, 937, 1990 |
| 人类 | 人类 | Chorionic, placental | Deoxyribonuclease I: 0.003% | HBSS | Egan, D., Grzegorczyk, V., Tricarico, K., Rueter, A.H., Olleman, W., and Marcotte, P.: Human Placental Chorionic Renin: Production, Purification and Characterization, Biochim Biophys Acta 965, 68, 1988 |
| 人类 | 人类, 女性 | Endometrial | Collagenase: 2% | RPMI 1640 | Rinehart, C., Lyn-Cook, B., and Kaufman, D.: Gland formation from human endometrial epithelial cells in vitro, In Vitro Cell Dev Biol 24 (10), 1037, 1988 |
| 人类 | 人类 | Placental | Trypsin: 0.25% | DMEM | Morrish, D., and Siy, O.: Critical Factors in Establishing Monolayer Cultures of Normal Human Placental Cells in Serum-Free Medium, Endocr Res 12 (3), 229, 1986 |
| 人类 | 人类 | Interna & corpus luteum | Pronase: 0.1% | Moscona's BSS | Marcus, G., Connor, L., Domingo, M., Tsang, B., Downey, B., and Ainsworth, L.:Enzymatic Dissociation of Ovarian and Uterine Tissues, Endocr Res 10, 151, 1984 |
| (包括 猪，牛) | Endometrium | ||||
| Ovarian | |||||
| Uterine | |||||
| 人类 | 人类,女性, 27-49 岁 | Epithelial | Trypsin: 0.125% | HBSS, CMF | Auersperg, N., Siemens, C.H., and Myrdal, S.E.: Human Ovarian Surface Epithelium In Primary Culture, In Vitro 20, 743, 1984 |
| Ovary | |||||
| 人类 | 人类 | Epithelial | Collagenase: 0.25% | See reference | Siegfried, J., Nelson, K., Martin, J., and Kaufman, D.: Histochemical Identification of Cultured Cells From Human Endometrium, In Vitro 20 (1), 25, 1984 |
| Stromal | |||||
| 人类 | 人类, 婴儿和新生儿 | Epithelial | Trypsin: 0.1% | HBSS | Lechner, J., Babcock, M., Marnell, M., Narayan, K., and Kaighn, M.: Normal Human Prostate Epithelial Cell Cultures, Methods Cell Biol 21, 195, 1980 |
| Prostate | |||||
| 人类 | 人类, 女性, 27 岁 | Smooth muscle, uterine | Trypsin: 0.05% | EBSS | Rifas, L., Fant, J., Makman, M., and Seifter, S.: The Characterization of Human Uterine Smooth Muscle Cells in Culture, Cell Tissue Res 196, 385, 1979 |
| 人类 | 人类 | Epithelial | Collagenase Type 1: 180 u/ml | DMEM | Kirk, D., King, R., Heyes, J., Peachey, L., Hirsch, P., and Taylor, W.: Normal Human Edometrium in Cell Culture, In Vitro 14 (8), 651, 1978 |
| Stromal | |||||
| 昆虫 | 果蝇 | Ovarian | Collagenase Type 2: 0.1% | Grace's | Ma, X., Wang, S., Do, T., Song, X., Inaba, M., Nishimoto, Y., Liu, L., Gao, Y., Mao, Y., Li, H., McDowell, W., Park, J., Malanowski, K., Peak, A., Perera, A., Li, H. et al.: Piwi is Required in Multiple Cell Types to Control Germline Stem Cell Lineage Development in the Drosophila Ovary., PLoS ONE 9, e90267, 2014 |
| 昆虫 | 果蝇, 胚胎 | Cardiac differentiating | Collagenase Type 1: 20 u/ml | Schneider | Salmand, P. Iche-Torres, M. and Perrin, L.: Tissue-Specific Cell Sorting from Drosophila Embryos: Application to Gene Expression Analysis.,Fly (Austin) Vol. 5,, , 261, |
| Trypsin: 0.25% | |||||
| Deoxyribonuclease I: 4 u/ml | |||||
| 小鼠 | 小鼠, 成年的 | Spermatogonial stem | Trypsin: 0.05% | DMEM | Martin, L. and Seandel, M.: Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-Term Adult Mouse SSC Lines., J Vis Exp 72, e50017, 2013 |
| Collagenase Type 1: 0.03% | |||||
| Deoxyribonuclease I: 80 u/ml | |||||
| 小鼠 | 小鼠 | Ovarian | Collagenase Type 4: 800 u/ml | HBSS | White, Y., Woods, D., Takai, Y., Ishihara, O., Seki, H. and Tilly, J.: Oocyte Formation by Mitotically Active Germ Cells Purified from Ovaries of Reproductive-Age Aomen., Nat Med 18, 413, 2012 |
| 小鼠 | 小鼠, 雌性 | Lymphocytes | Collagenase: 450 u/ml | RPMI | Jiang, J. and Kelly, K.: Isolation of Lymphocytes from Mouse Genital Tract Mucosa.,J Vis Exp 67, e4391, 2012 |
| 小鼠 | 小鼠 | Vaginal smooth muscle | Collagenase Type 2: 175 u/ml | DMEM/F12 | Venkataraman, L., Lenis, A., Couri, B., Damaser, M. and Ramarurthi, A: Induced Regenerative Elastic Matrix Repair in LOXL 1 Knockout Mouse Cell Cultures: Towards Potential Therapy for Pelvic Organ Prolapse, J Tissue Sci Eng 3, 3, 2012 |
| Elastase: 0.025% | |||||
| 小鼠 | 小鼠, 雄性 | Testis, meiotic | Collagenase Type 1: 120 u/ml | Gey's BSS | Getun, I., Torres, B. and Bois, P.: Flow Cytometry Purification of Mouse Meiotic Cells., J Vis Exp 50, 2602, 2011 |
| Deoxyribonuclease I: 0.001% | |||||
| Trypsin: 0.1% | |||||
| 小鼠 | 小鼠 | Uterine stomal | Neutral Protease: 0.6% | DMEM/F12 | Chen, L., Belton, R. and Nowak, R.: Basigin-Mediated Gene Expression Changes in Mouse Uterine Stromal Cells During Implantation., Endocrinology 150, 966, 2009 |
| Pancreatin: 0.25% | |||||
| Collagenase Type 3: 0.05% | |||||
| 小鼠 | 小鼠 | Germ cells | Collagenase Type 1: 100 u/ml | HBSS | Breault, D., Min, I., Carlone, D., Farilla, L., Ambruzs, D., Henderson, D., Algra, S., Montgomery, R., Wagers, A. and Hole, N.: Generation of mTert-GFP Mice as a Model to Identify and Study Tissue Progenitor Cells., Proc Natl Acad Sci U S A 105, 10420, 2008 |
| 小鼠 | 小鼠, 胚胎 | Hematopoietic stem cells | Collagenase: 0.1% | PBS | Gekas, C., Rhodes, K. and Mikkola, H.: Isolation and Analysis of Hematopoietic Stem Cells from the Placenta., J Vis Exp 16, e742, 2008 |
| 小鼠 | 小鼠, 6 天 | Sertoli | Collagenase Type 2: 500 u/ml | DMEM | Nalbandian Angele, Dettin Luis, Dym Martin, Ravindranath Neelakanta:Expression of vascular endothelial growth factor receptors during male germ cell differentiation in the mouse, Biol Reprod 69, 985-94, 2003 |
| Hyaluronidase: 0.1% | |||||
| Deoxyribonuclease I: .0005% | |||||
| 小鼠 | 小鼠, 成年的, 新生儿与胎儿 | Testicular cells | Collagenase Type 1: 0.1% | DMEM/F-12 | O'Shaughnessy PJ, Fleming LM, Jackson G, Hochgeschwender U, Reed P, Baker PJ: Adrenocorticotropic hormone directly stimulates testosterone production by the fetal and neonatal mouse testis, Endocrinology 144, 3279-84, 2003 |
| 小鼠 | 小鼠, 雄性, 3.5 个月大 | Leydig | Collagenase: 0.06% | Medium E 199 | Faldikova, L., Diblikova, I., Canderle, J., Zraly, Z., Veznik, Z., and Sulcova, A.: Effects of Nutrition, Social Factors and Chronic Stress on the Mouse Leydig Cell Testosterone Production, Vet Med 46 (6), 160, 2001 |
| 小鼠 | 小鼠, 雄性, 12-14-周大 | Seminiferous tubules | Trypsin: 0.05% | DMEM | Lin, Q., Sirotkin, A., and Skoultchi, A.: Normal Spermatogenesis in Mice Lacking the Testis-Specific Linker Histone H1t, Mol Cell Biol 20 (6), 2122, 2000 |
| 小鼠 | 小鼠, 6-12 天 | Oocyte-granulosa | Collagenase Type 1: 0.1% | Waymouth | Eppig JJ, O'Brien MJ: Development in vitro of mouse oocytes from primordial follicles, Biol Reprod 54, 197-207, 1996 |
| Deoxyribonuclease I: 0.02% | |||||
| 小鼠 | 小鼠, 雌性,6-8 周 | Uterine | Trypsin: 0.2% | HBSS | Ghosh, D., Danielson, K., Alston, J., Heyner, S.: Functional Differentiation of Mouse Uterine Epithelial Cells Grown On Collagen Gels Or Reconstituted Basement Membranes, In Vitro Cell Dev Biol 27, 713, 1991 |
| 小鼠 | 小鼠, 雌性, 21 天, 10-12 克 | vaginal epithelial | Collagenase Type 3: 38 u/ml | DMEM | Tsai, P., Uchima, F., Hamamoto, S., and Bern, H.: Proliferation and Differentiation of Prepubertal Mouse Vaginal Epithelial Cells In Vitro and the Specificity of Estrogen-Induced Growth Retardation, In Vitro Cell Dev Biol 27A, 461, 1991 |
| 小鼠 | 小鼠, CF1, 雌性 | Cumulus, one-cell embryos | Hyaluronidase: 0.1% | PBS, CMF | Spindle, A.: In vitro Development of One-Cell Embryos from Outbred Mice: Influence of Culture Medium Composition, In Vitro Cell Dev Biol 26, 151, 1990 |
| 小鼠 | 小鼠 (白变种实验室老鼠CRGL), 雄性, 2-3 个月 | Epithelial, prostate gland | Hyaluronidase: 0.1% | Medium 199 | Turner, T., Bern, H., Young,P., and Cunha, G.: Serum-Free Culture of Enriched Mouse Anterior and Ventral Prostatic Epithelial Cells in Collagen Gel, In Vitro Cell Dev Biol 26, 722, 1990 |
| 小鼠 | 小鼠 (C57B/T), 胎儿, 16-17 天大 | Prostate | Trypsin: 1.0% | HBSS/ DMEM | Thompson, T.C., Southgate, J., Kitchener, G., and Land, H.: Multistage Carcinogenesis Induced by ras and myc Oncogenes in a Reconstituted Organ, Cell56, 917, 1989 |
| 小鼠 | 小鼠, outbred, CD-1, 21-23 天s old | Epithelial | Trypsin: 0.5% | Medium 199 | Tomooka, Y., DiAugustine, R., and McLachlan, J.: Proliferation of Mouse Uterine Epithelial Cells in Vitro, Endocrinology 118 (3), 1011, 1986 |
| 小鼠 | 小鼠, 白变种实验室老鼠 | Epithelial | Trypsin: 1% | DMEM | Bigsby, R., Cooke, P., and Cunha, G.: A Simple Efficient Method For Separating Murine Uterine Epithelial and Mesenchymal Cells, Am J Physiol 251, E630, 1986 |
| Mesencymal | |||||
| 小鼠 | 小鼠, 白变种实验室老鼠Crg, 40 天大 | Epithelial | Trypsin: 1% | Medium 199 | Cooke, P., Uchima, F., Fujii, D., Bern, H., and Cunha, G.: Restoration of Normal Morphology and Estrogen Responsiveness in Cultured Vaginal and Uterine Epithelia Transplanted with Stroma, Proc Natl Acad Sci U S A 83, 2109, 1986 |
| 小鼠 | 小鼠, 雄性, 10-13 周 | Leydig | Deoxyribonuclease I: 0.001% | Medium 199 w/ BSA | Stalvcey, J. and Payne, A.: Luteinizing Hormone Receptors and Testosterone Production in Whole Testes and Purified Leydig Cells from the Mouse: Differences among Inbred Strains, Endocrinology 112, 1696, 1983 |
| Testis | |||||
| 小鼠 | 小鼠 (白变种实验室老鼠Crgl), 雌性, 50 - 60 天 | Epithelial, vagina | Collagenase Type 3: 0.1% | HBSS | Iguchi, T., Uchima, F.A., Ostrander, P., and Bern, H.: Growth of Normal Mouse Vaginal Epithelial Cells in and on Collagen Gels, Proc Natl Acad Sci U S A 80, 3743, 1983 |
| 绵羊 | 羊 | Epithelial | Collagenase: 125 - 190 u/ml | DMEM | Salamonsen, L., Sum O, W., Doughton, B., and Findlay, J.: The Effects of Estrogen and Progesterone In Vivo on Protein Synthesis and Secretion by Cultured Epithelial Cells from Sheep Endometrium, Endocrinology 117 (5), 2148, 1985 |
| 猪 | 猪, 雄性, 8 天 | Seminiferous epithelial cells | Collagenase: 0.15% | DMEM/F12 | Dirami G, Ravindranath N, Pursel V, Dym M: Effects of stem cell factor and granulocyte macrophage-colony stimulating factor on survival of porcine type A spermatogonia cultured in KSOM, Biol Reprod 61, 225-30, 1999 |
| Deoxyribonuclease I: .0001% | |||||
| Hyaluronidase: 0.15% | |||||
| Trypsin: 0.05% | |||||
| 猪 | 猪, 雌性 | Corpus Leuteum | Collagenase Type 4: 600 u/ml | Medium 199 | Ciereszko, R., Petroff, B., Ottobre, A., Guan, Z., Stokes, B., and Ottobre, J.:Assessment of the Mechanism by Which Prolactin Stimulates Progesterone Production by Early Corpora Lutea of Pigs, J Endocrinol 159, 201, 1998 |
| 猪 | 猪 (包括牛，人类) | Interna & corpus luteum | Pronase: 0.1% | Moscona's BSS | Marcus, G., Connor, L., Domingo, M., Tsang, B., Downey, B., and Ainsworth, L.:Enzymatic Dissociation of Ovarian and Uterine Tissues, Endocr Res 10, 151, 1984 |
| Endometrium | |||||
| Ovarian | |||||
| Uterine | |||||
| 猪 | 猪, 3-4 周 | Leydig | Trypsin: 0.0003% | Lebovitz L-15 Medium | Mather, J., Saez, J., and Haour, F.: Regulaton of Gonadotropin Receptors and Steroidogenesis in Cultured Porcine Leydig Cells, Endocrinology 110, 933, 1982 |
| Testis | |||||
| 兔 | 兔, 新西兰, 3 个月 | Testicular germ | Collagenase Type 1: 0.1% | HBSS | Kubota, H., Wu, X., Goodyear, S., Avarbock, M. and Brinster, R.: Glial Cell Line-Derived Neurotrophic Factor and Endothelial Cells Promote Self-Renewal of Rabbit Germ Cells with Spermatogonial Stem Cell Properties., FASEB J 25, 2604, 2011 |
| Trypsin: 0.25% | |||||
| Deoxyribonuclease I: 0.7% | |||||
| 兔 | 兔, 新西兰 白, 4-6 个月大, | Ovarian | Collagenase Type 1: 300 u/ml | HBSS | Setrakian, S., Oliveros-Saunders, B., and Nicosia, S.: Growth Stimulation of Ovarian and Extraovarian Mesothelial Cells by Corpus Luteum Extract, In Vitro Cell Dev Biol 29A, 879, 1993 |
| Mesothelial | |||||
| 兔 | 兔, 新西兰白, 成年的, 雌性 (非妊娠的), | Myocytes, uterine | Deoxyribonuclease I: 200 礸/ml | HBSS-HEPES buffer | Phillippe, M., Saunders, T., and Bangalore, S.: Alpha-1,Alpha-2,and Beta Adrenergic Signal Transduction in Cultured Uterine Myocytes, In Vitro Cell Dev Biol26, 369, 1990 |
| 3-4公斤 | |||||
| 兔 | 兔, 新西兰, 成年的, 雌性, 4-5公斤 | Ovarian surface epithelial and peritoneal mesothelial | Collagenase Type 1: 300 IU/ml (280 IU/mg) | HBSS, CMF | Piquette, G., and Timms, B.: Isolation and Characterization of Rabbit Ovarian Surface Epithelium, Granulosa Cells, and Peritoneal Mesothelium in Primary Culture,In Vitro Cell Dev Biol 26, 471, 1990 |
| 兔 | 兔, 混合品种 | Endometrial epithelial | Collagenase Type 1: 0.005% | DMEM | Mulholland, J., Winterhager, E., and Beier, H.: Changes in Proteins Synthesized by Rabbit Endometrial Epithelial Cells Following Primary Culture, Cell Tissue Res 252, 123, 1988 |
| 兔 | 兔, 新西兰 白, 成年的, 3-4 公斤 | Myometrial | Trypsin: 0.02%, 0.03%, 0.0375% | HBSS | Boulet, A., and Fortier, M.: Preparation and Characterization of Rabbit Myometrial Cells in Primary Culture: Influence of Estradiol and Progesterone Treatment, In Vitro Cell Dev Biol 23, 93, 1987 |
| 兔 | 兔, 新西兰白发情的, 雌性, 4-5 个月 | Mesothelial and surface epithelial | Trypsin: 0.125%-0.5% | Medium 199 | Nicosia, S., Johnson, J., and Streibel, E.: Isolation and Ultrastructure of Rabbit Ovarian Mesothelium(Surface Epithelium), Int J Gynecol Pathol 3, 348, 1984 |
| Ovaries | |||||
| 大鼠 | SD大鼠, 350-450 克 | Leydig cells | Collagenase: .05-0.1% | Medium 199 | Sharma RS, Pal PC, Rajalakshmi M.: Isolation and Culture of Leydig Cells from Adult Rats, Indian J Clinical Biochem 21, 27, 2006 |
| 大鼠 | SD大鼠, 成年的, 雄性, 8 - 10 周 | Seminiferous tubules | Trypsin: 0.05% | Krebs-Ringer bicarbonate buffer | Abou-Haila, A., and Tulsiani, D.: Acid Glycohydrolases in Rat Spermatocytes, Spermatids and Spermatozoa: Enzyme Activities, Biosynthesis and Immunolocalization, Biol Proced Online 3 (1), 35, 2001 |
| (see reference) | |||||
| 大鼠 | SD大鼠, 未成熟的 | Ovary | Collagenase Type 1: 144 u/ml | McCoy's | Ando, M., Kol, S., Irahara, M., Sirois, J., and Adashi, E.: Non-Steroidal Anti-Inflammatory Drugs (NSAIDS) Block the Late, Prostanoid-Dependent/Ceramide-Independent Component of Ovarian IL-1 Action: Implications for the Ovulatory Process, Mol Cell Endocrinol 157, 21, 1999 |
| 大鼠 | Han-Wistar大鼠, 成年的, 雄性, 250 克 | Testes | Trypsin: 0.1% | Medium 199 w/ Hank's salts | Leckie, C., Welberg, L., and Seckl, J.: 11B-Hydroxysteroid Dehydrogenase is a Predominant Reductase in Intact Rat Leydig Cells, J Endocrinol 159, 233, 1998 |
| 大鼠 | SD大鼠, 雌性, 未成熟的, 26 天 | Corpus luteum | Neutral Protease: 2.4 u/ml Deoxyribonuclease: 200u/ml | Serum-free medium | lieria, C., Ou, J., Sugino, N., Ferguson, S., and Gibori, G.: The Expression of Interleukin-6 in the Pregnant Rat Corpus Luteum and Its Regulation by Progesterone and Glucocorticoid, Endocrinology 139 (8), 3597, 1998 |
| (包括 大鼠, 妊娠和假孕) | (see reference) | ||||
| 大鼠 | SD大鼠, 25 天 | Ovarian theca-interstitial | Collagenase Type 1: 0.5% | Medium 199 | Duleba AJ, Spaczynski RZ, Olive DL, Behrman HR: Effects of insulin and insulin-like growth factors on proliferation of rat ovarian theca-interstitial cells, Biol Reprod 56, 891-7, 1997 |
| Deoxyribonuclease I: 0.02% | |||||
| 大鼠 | SD大鼠, 雄性, 90 天 | Sertoli | Hyaluronidase: 0.1% | DMEM | Onoda, M. and Djakiew, D.: Pachytene Spermatocyte Protein(s) Stimulate Sertoli Cells Grown in Bicameral Chambers: Dose-Dependent Secretion of Ceruloplasmin, Sulfated Glycoprotein-1, Sulfated Glycoprotein-2, and Transferrin, In Vitro Cell Dev Biol 27, 215, 1991 |
| 大鼠 | SD大鼠, 雄性 | Leydig | Trypsin: 0.02% | DMEM | Abayasekara, D., Kurlak, L., Band, A., Sullivan, M., and Cooke, B.: Effect of Cell Purity, Cell Concentration, and Incubation Conditions on Rat Testis Leydig Cell Steroidogenesis, In Vitro Cell Dev Biol 27, 253, 1991 |
| 大鼠 | 大鼠, 新生的 | Uterine | Trypsin: 1% | HBSS | Branham, W., Lyn-Cook, B., Andrews, A., McDaniel, M., Sheehan, D.: Growth of Neonatal Rat Uterine Luminal Epithelium on Extracellular Matrix, In Vitro Cell Dev Biol27, 442, 1991 |
| 大鼠 | SD大鼠, 雄性, 120-160 克 | 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 |
| Adrenal | |||||
| 大鼠 | Wistar大鼠, 成年的 | Uterine | Trypsin: 0.5% | PBS | Pampfer, S., Vanderheyden, I., Michiels, B., and DeHertogh, R.: Co-Culture of Two-Cell Rat Embryos on Cell Monolayers, In Vitro Cell Dev Biol 26, 944, 1990 |
| 大鼠 | SD大鼠, 10 天 | Sertoli cells | Collagenase Type 2: 500 u/ml | PBS | Hadley MA, Weeks BS, Kleinman HK, Dym M: Laminin promotes formation of cord-like structures by Sertoli cells in vitro, Dev Biol 140, 318-27, 1990 |
| Deoxyribonuclease I: 0.0005% | |||||
| Hyaluronidase: 0.1% | |||||
| 大鼠 | 大鼠, 未成熟的 | Luteal, ovaries | Collagenase: 0.3% | McCoy's | Rajan, V. and Menon, K.: Differential Uptake and Metabolism of Free and Esterified Cholesterol from High-density Lipoproteins in the Ovary, Biochim Biophys Acta 959, 206, 1988 |
| 大鼠 | 大鼠, 雄性, 20 天 | Sertoli | Trypsin: 0.15% | (see reference) | Skinner, M., Fetterolf, P., and Anthony, C.: Purification of a Paracrine Factor, P-Mod-S,Produced by Testicular Peritubular Cells That Modulates Sertoli Cell Function,J Biol Chem 263, 2884, 1988 |
| 大鼠 | SD大鼠, 雌性 | Luminal epithelial | Trypsin: 0.5% | HBSS | Glasser, S., Julian, J., Decker, G., Tang, J., and Carson, D.: Development of Morphological and Functional Polarity in Primary Cultures of Immature Rat Uterine Epithelial Cells, J Cell Biol 107 (6), 2409, 1988 |
| 大鼠 | Wistar大鼠, 处女, 雌性, 250 克 | Vaginal epithelial | Trypsin: 0.5% | PBS | Conti, C., and Tasat, D.: Regulation of Cultured Rat Vaginal Epithelial Cells By 17B-Estradiol and Progesterone, J Steroid Biochem 24 (3), 747, 1986 |
| 大鼠 | SD大鼠, 雌性, 21 天 | Luteal, ovaries | Deoxyribonuclease I: 0.0004% | McCoy's | Rajan, V. and Menon, K.: Involvement of Microtubules in Lipoprotein Degradation and Utilization for Steroidogenesis in Cultured Rat Luteal Cells, Endocrinology 117, 2408, 1985 |
| 大鼠 | SD大鼠, 雄性, 10 天 | Sertoli | Trypsin: 0.025% | DMEM | Hadley, M., Byers, S., Suarez-Quian, C., Kleinman, H., and Dym, M.: Extracellular Matrix Regulates Seratoli Cell Differentiation, Testicular Cord Formation and germ cell development in vitro, J Cell Biol 101, 1511, 1985 |
| 大鼠 | Holtzman大鼠, 雌性, 未成熟的 | Uterine | Deoxyribonuclease I: 0.025% | DMEM buffered with HEPES | Kassis, J., Walent, J., and Gorski, J.: Estrogen Receptors in Rat Uterine Cell Cultures: Effects of Medium on Receptor Concentration, Endocrinology 115, 762, 1984 |
| 大鼠 | SD大鼠, 成年的,雄性, 50-70 天 | Testicular | Deoxyribonuclease I: 10 礸/ml | HEPES | Hsueh, A., Bambino, T., Zhuang, L., Welsh, T., and Ling, N.: Mechanism of the Direct Action of Gonadotropin Releasing Hormone and Its Antagonist on Androgen Biosynthesis by Cultured Rat Testicular Cells, Endocrinology 112, 1653, 1983 |
| 大鼠 | SD大鼠, 雌性, 假妊娠的, 21 天 | Luteal, ovaries | Hyaluronidase: 0.1% | EBSS | Rajendran, K., Hwang, J., and Menon, K.: Binding, Degradation an Utilization of Plasma High Density and Low Density Lipoproteins for Progesterone Production In Cultured Rat Luteal Cells, Endocrinology 112, 1746, 1983 |
| 大鼠 | SD大鼠, 4-6 天, 雄性 | Sertolli, seminiforous tubules | Collagenase: 0.03% | Serum-free medium | Rich, K., Bardin, C., Gunsalus, G., and Mather, J.: Age-Dependent Pattern of Androgen-Binding Protein Secretion from Rat Sertoli Cells in Primary Culture,Endocrinology 113, 2284, 1983 |
| 大鼠 | SD大鼠, 雌性, 26 天 | Luteal | Deoxyribonuclease I: 0.0004% | Medium 199 | Azhar, S. and Reaven, E.: Effect of Antimicrotubule Agents on Microtubules and Steroidogenesis in Luteal Cells, Am J Physiol 243, E380, 1982 |
| 大鼠 | SD大鼠, 雄性 | Leydig | Collagenase: 0.1% | Krebs Ringer bicarbonate buffer | Ramachandran, J., and Sairam, M.R.: The Effects Of Interstitial Cell-Stimulating Hormone, Its Subunits,and Recombinants on Isolated Rat Leydig Cells, Arch Biochem Biophys 167, 294, 1975 |
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