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生殖組織消化手冊

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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, 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

心臟

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。

 

生殖組織消化手冊

 

物種詳細來源細胞名稱消化用酶培養基參考文獻
Corpus leuteal cellsCollagenase Type 4: 420 u/mlM-199Levy 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, 雌性LeutealCollagenase Type 1: 0.2%Ham's F-12Tsang 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
牛犢,中晚期妊娠FibroblastsCollagenase: 0.1%Medium 199Coplen, 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
EpithelialCollagenase Type 2: 0.1%DMEM/EBSSMunson, L., Chandler, S., and Schlafer, D.: Long-Term Culture of Bovine Trophoblastic Cells, J Tiss Cul Meth 11 (3), 123, 1988
Endometrial
牛 Interna & corpus luteumPronase: 0.1%Moscona's BSSMarcus, 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
Amniotic membrane MSCTrypsin: 0.25%LG-DMEMPark, 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, 家雞,20-30 周Primary folliclesTrypsin: 0.15%Dulbecco's phosphate buffered salineDu 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OocytesCollagenase: 0.2%Barth's solutionChatzidaki, 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OocytesCollagenase: 0.2%see referenceMruk, 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OocytesCollagenase: 0.5%Barth's solution, CFCohen, 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OocytesCollagenase Type 1: 0.2%see referencePannaccione 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OocytesCollagenase Type 1: 0.2%CF MediumAlagem, 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OocytesCollagenase 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OocytesCollagenase: 0.1%Barth's solution, CFKarkanias, 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OocytesCollagenase: 0.2%CF MediumMoriarty, 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,中國的OvaryTrypsin: 5%Dialyzed fetal calf serum, 10% and 0.5M MethotrexateWallis, 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 formingCollagenase Type 1: 0.1%HBSSPa, 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 epithelialPancreatin: 0.34%HBSSPa, 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 stemCollagenase Type 1: 0.4%DMEM/F12Shalini, 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 stemCollagenase Type 2: 10%CMF-DPBSSteigman, 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DecidualCollagenase: 0.25%DMEM/F12Lockwood, 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 StromalTrypsin: 0.1-0.25%HBSS RPMISpessotto, 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 cellsTrypsin: 2.5%DMEM/F-12Teng 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 epithelialPancreatin: 0.34%HBSSMeter, 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 cellsCollagenase Type 3: 0.03%DMEM/F-12Chan 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 placentalCollagenase Type 1: 0.2%DMEMWang 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 fibroblastTrypsin: 0.125%PBSSun 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 cellsPancreatin: 0.34%HBSSFahey 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EndothelialCollagenase Type 1/2/4: 0.1%DMEMOberlin, 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MesothelialCollagenase Type 1: 0.1%Eagle's MEMWitz 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, embryonicNeutral Protease: 0.01% - 0.02%DMEMZhang, 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 cellsCollagenase Type 1: 0.2%HBSSArnold, 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 villiTrypsin: see referenceHBSSYusuf 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 cellsCollagenase Type 1: 0.2%McCoys mediumNikitenko 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 cellsCollagenase Type 2: 0.2%PBSGargett 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 cellsCollagenase Type 3: 45 u/mlDMEM/F-12Zhang 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 cellsCollagenase Type 1: 0.3%PBSRunesson 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 cellsCollagenase Type 2: 0.25%PBSFriden 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 cellsCollagenase Type 2: 0.25%PBSFriden 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FolliclesCollagenase Type 2: 0.025-0.1%EBSSHovatta 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PlacentalDeoxyribonuclease I: 0.04%PSSBradbury., 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 cellsCollagenase: 4000 u/mlDMEM/F-12Huang 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 endometrialCollagenase Type 3: 45 u/mlDMEM/Ham's F12Zhang 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 tubeCollagenase Type 1: 1%Medium 199Takeuchi, 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, placentalTrypsin: 0.25%EBSS, CMFBranchaud, 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, placentalTrypsin: 0.25%PBSJie, 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, placentalDeoxyribonuclease I: 0.003%HBSSEgan, 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EndometrialCollagenase: 2%RPMI 1640Rinehart, 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PlacentalTrypsin: 0.25%DMEMMorrish, 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 luteumPronase: 0.1%Moscona's BSSMarcus, 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EpithelialTrypsin: 0.125%HBSS, CMFAuersperg, 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EpithelialCollagenase: 0.25%See referenceSiegfried, 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EpithelialTrypsin: 0.1%HBSSLechner, 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, uterineTrypsin: 0.05%EBSSRifas, 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EpithelialCollagenase Type 1: 180 u/mlDMEMKirk, 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OvarianCollagenase Type 2: 0.1%Grace'sMa, 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 differentiatingCollagenase Type 1: 20 u/mlSchneiderSalmand, 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 stemTrypsin: 0.05%DMEMMartin, 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OvarianCollagenase Type 4: 800 u/mlHBSSWhite, 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LymphocytesCollagenase: 450 u/mlRPMIJiang, J. and Kelly, K.: Isolation of Lymphocytes from Mouse Genital Tract Mucosa.,J Vis Exp 67, e4391, 2012
小鼠小鼠Vaginal smooth muscleCollagenase Type 2: 175 u/mlDMEM/F12Venkataraman, 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, meioticCollagenase Type 1: 120 u/mlGey's BSSGetun, 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 stomalNeutral Protease: 0.6%DMEM/F12Chen, 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 cellsCollagenase Type 1: 100 u/mlHBSSBreault, 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 cellsCollagenase: 0.1%PBSGekas, 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SertoliCollagenase Type 2: 500 u/mlDMEMNalbandian 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 cellsCollagenase Type 1: 0.1%DMEM/F-12O'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LeydigCollagenase: 0.06%Medium E 199Faldikova, 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 tubulesTrypsin: 0.05%DMEMLin, 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-granulosaCollagenase Type 1: 0.1%WaymouthEppig 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UterineTrypsin: 0.2%HBSSGhosh, 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 epithelialCollagenase Type 3: 38 u/mlDMEMTsai, 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 embryosHyaluronidase: 0.1%PBS, CMFSpindle, 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 glandHyaluronidase: 0.1%Medium 199Turner, 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ProstateTrypsin: 1.0%HBSS/ DMEMThompson, 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 oldEpithelialTrypsin: 0.5%Medium 199Tomooka, 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EpithelialTrypsin: 1%DMEMBigsby, 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EpithelialTrypsin: 1%Medium 199Cooke, 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LeydigDeoxyribonuclease I: 0.001%Medium 199 w/ BSAStalvcey, 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, vaginaCollagenase Type 3: 0.1%HBSSIguchi, 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EpithelialCollagenase: 125 - 190 u/mlDMEMSalamonsen, 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, 雄性, 8 天Seminiferous epithelial cellsCollagenase: 0.15%DMEM/F12Dirami 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, 雌性Corpus LeuteumCollagenase Type 4: 600 u/mlMedium 199Ciereszko, 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 (包括牛,人類)Interna & corpus luteumPronase: 0.1%Moscona's BSSMarcus, 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, 3-4 周LeydigTrypsin: 0.0003%Lebovitz L-15 MediumMather, J., Saez, J., and Haour, F.: Regulaton of Gonadotropin Receptors and Steroidogenesis in Cultured Porcine Leydig Cells, Endocrinology 110, 933, 1982
Testis
兔, 新西蘭, 3 個月Testicular germCollagenase Type 1: 0.1%HBSSKubota, 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, 新西蘭 白, 4-6 個月大,Ovarian Collagenase Type 1: 300 u/mlHBSSSetrakian, 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, 新西蘭白, 成年的, 雌性 (非妊娠的), Myocytes, uterineDeoxyribonuclease I: 200 礸/mlHBSS-HEPES bufferPhillippe, 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, 新西蘭, 成年的, 雌性, 4-5公斤Ovarian surface epithelial and peritoneal mesothelialCollagenase Type 1: 300 IU/ml (280 IU/mg)HBSS, CMFPiquette, 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, 混合品種Endometrial epithelialCollagenase Type 1: 0.005%DMEMMulholland, 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, 新西蘭 白, 成年的, 3-4 公斤MyometrialTrypsin: 0.02%, 0.03%, 0.0375%HBSSBoulet, 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, 新西蘭白發情的, 雌性, 4-5 個月Mesothelial and surface epithelialTrypsin: 0.125%-0.5%Medium 199Nicosia, 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 cellsCollagenase: .05-0.1%Medium 199Sharma 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 tubulesTrypsin: 0.05%Krebs-Ringer bicarbonate bufferAbou-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OvaryCollagenase Type 1: 144 u/mlMcCoy'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TestesTrypsin: 0.1%Medium 199 w/ Hank's saltsLeckie, 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 luteumNeutral Protease: 2.4 u/ml Deoxyribonuclease: 200u/mlSerum-free mediumlieria, 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-interstitialCollagenase Type 1: 0.5%Medium 199Duleba 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SertoliHyaluronidase: 0.1%DMEMOnoda, 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LeydigTrypsin: 0.02%DMEMAbayasekara, 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UterineTrypsin: 1%HBSSBranham, 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LeydigCollagenase Type 2: 0.03% (adrenal)Krebs Ringer bicarbonate bufferNg, 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UterineTrypsin: 0.5%PBSPampfer, 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 cellsCollagenase Type 2: 500 u/mlPBSHadley 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, ovariesCollagenase: 0.3%McCoy'sRajan, 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SertoliTrypsin: 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 epithelialTrypsin: 0.5%HBSSGlasser, 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 epithelialTrypsin: 0.5%PBSConti, 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, ovariesDeoxyribonuclease I: 0.0004%McCoy'sRajan, 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SertoliTrypsin: 0.025%DMEMHadley, 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UterineDeoxyribonuclease I: 0.025%DMEM buffered with HEPESKassis, 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TesticularDeoxyribonuclease I: 10 礸/mlHEPESHsueh, 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, ovariesHyaluronidase: 0.1%EBSSRajendran, 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 tubulesCollagenase: 0.03%Serum-free mediumRich, 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LutealDeoxyribonuclease I: 0.0004%Medium 199Azhar, 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LeydigCollagenase: 0.1%Krebs Ringer bicarbonate bufferRamachandran, 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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