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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，但是對其組織消化和細(xì)胞收集的作用機(jī)制卻沒有全面的理解。 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)選擇或基于過去的經(jīng)驗，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)具體實驗?zāi)康男拚桨敢垣@得更好的實驗結(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(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、孵育時間
通常前四個原因沒辦法選擇，而剩下的原因一般由經(jīng)驗決定以獲取zui適實驗結(jié)果。
通過查閱文獻(xiàn)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(shù)據(jù)搞得一頭霧水。這些巨大差異來源于組織胞外基質(zhì)的復(fù)雜而動態(tài)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(zhì)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(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)胞收集知識和方法，描述了標(biāo)準(zhǔn)的實驗步驟， 以便于制定一個*的細(xì)胞分離方案，同時列舉了一些相關(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(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型的消化。
生殖組織消化手冊
| 物種 | 詳細(xì)來源 | 細(xì)胞名稱 | 消化用酶 | 培養(yǎng)基 | 參考文獻(xiàn) |
| 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 | 牛犢,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 |
| (包括豬，人類) | 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% | |||||
| 雞 | 雞, 家雞,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 |
| 馬 | 馬，中國的 | 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 |
| 人類 | 人類 | 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% | |||||
| 人類 | 人類 | 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% | |||||
| 人類 | 人類 | 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% | |||||
| 人類 | 人類 | 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 | |||||
| 人類 | 人類, 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 | |||||
| 人類 | 人類 | 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% | |||||
| 人類 | 人類 | 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% | |||||
| 人類 | 人類, 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% | |||||
| 人類 | 人類, 女性, 34-51 歲 | 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% | |||||
| 人類 | 人類 | 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% | |||||
| 人類 | 人類 | 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% | |||||
| 人類 | 人類 | 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% | |||||
| 人類 | 人類,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 | |||||
| 人類 | 人類, 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% | |||||
| 人類 | 人類 | 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 |
| 人類 | 人類, 20-40 歲 | 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 |
| 人類 | 人類 | 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 | |||||
| 人類 | 人類 | 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 |
| 人類 | 人類, 女性, 20-40 歲 | 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% | |||||
| 人類 | 人類,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% | |||||
| 人類 | 人類 | 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% | |||||
| 人類 | 人類 | 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% | |||||
| 人類 | 人類, 女性, 25-45 歲 | 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% | |||||
| 人類 | 人類, 女性, 25-41 歲 | 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 |
| 人類 | 人類 | 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% | |||||
| 人類 | 人類, 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 |
| 人類 | 人類 | 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% | |||||
| 人類 | 人類 | 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 |
| 人類 | 人類 | 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 |
| 人類 | 人類 | 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 |
| 人類 | 人類 | 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 |
| 人類 | 人類, 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 |
| 人類 | 人類 | 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 |
| 人類 | 人類 | 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 豬，牛) | Endometrium | ||||
| Ovarian | |||||
| Uterine | |||||
| 人類 | 人類,女性, 27-49 歲 | 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 | |||||
| 人類 | 人類 | 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 | |||||
| 人類 | 人類, 嬰兒和新生兒 | 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 | |||||
| 人類 | 人類, 女性, 27 歲 | 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 |
| 人類 | 人類 | 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 | |||||
| 昆蟲 | 果蠅 | 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 |
| 昆蟲 | 果蠅, 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, 新生兒與胎兒 | 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 個月大 | 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 (白變種實驗室老鼠CRGL), 雄性, 2-3 個月 | 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), 胎兒, 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, 白變種實驗室老鼠 | 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, 白變種實驗室老鼠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 (白變種實驗室老鼠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 |
| 綿羊 | 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 |
| 豬 | 豬, 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% | |||||
| 豬 | 豬, 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 |
| 豬 | 豬 (包括牛，人類) | 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 | |||||
| 豬 | 豬, 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, 新西蘭, 3 個月 | 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, 新西蘭 白, 4-6 個月大, | 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, 新西蘭白, 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, 新西蘭, 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, 混合品種 | 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, 新西蘭 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, 新西蘭白發(fā)情的, 雌性, 4-5 個月 | 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, 處女, 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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