眼组织消化手册

　更新时间：2016-02-16　点击量：705

世界*实验材料供应商 Worthington Biochemical 正式授权上海起发为其中国代理， Worthington Biochemical 在一直是行业的*,一直为广大科研客户提供zui为的产品和服务,上海起发一直秉承为中国科研客户带来的产品，的服务，签约 Worthington Biochemical 就是为了给广大科研客户带来更加完善的产品和服务,您的满意将是我们zui大的收获

Worthington Biochemical 中国代理, Worthington Biochemical 上海代理， Worthington Biochemical 北京代理，Worthington Biochemical 广东代理， Worthington Biochemical 江苏代理Worthington Biochemical 湖北代理,Worthington Biochemical 天津,Worthington Biochemical 黑龙江代理,Worthington Biochemical 内蒙古代理,Worthington Biochemical 吉林代理,Worthington Biochemical 福建代理, Worthington Biochemical 江苏代理, Worthington Biochemical 浙江代理,Worthington Biochemical 四川代理,

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。

眼组织消化手册

物种	详细来源	细胞名称	消化用酶	培养基	参考文献
牛	牛	Pericyte	Collagenase: 0.2%	DMEM	Bryan, B. and D'Amore, P.: Pericyte Isolation and USe is Endothelial/Pericyte Coculture Models, Meth Enzymol 443, 315, 2008
牛	牛	Microvascular endothelial	Collagenase/Dispase: 0.1%	MEM	Bowman, P., Betz, A., and Goldstein, G.: Primary Culture of Microvascular Endothelial Cells From Bovine Retina, In Vitro 18 (7), 626, 1982
鸡	小鸡, 胚胎, 6 天	Retinal cells	Trypsin: 0.005%	DMEM/F12	Jacob Vanessa, Rothermel Andrae, Wolf Peter, Layer Paul G: Rhodopsin, violet and blue opsin expressions in the chick are highly dependent on tissue and serum conditions, Cell Death Differ 180, 159-68, 2005
鸡	小鸡, 胚胎, 6 天	Retinal cells	Deoxyribonuclease I: 0.005%	DMEM/F12
鸡	小鸡胚胎	Corneal epithelia	Collagenase: 0.08%	HBSS	Reenstra Wende R, Orlow Daniel L, Svoboda Kathy K H: ECM-stimulated signaling and actin reorganization in embryonic corneal epithelia are Rho dependent, Inv Ophthal Visual Sci 43, 3181-9, 2002
鸡	小鸡胚胎	Corneal epithelia	Trypsin: 0.08%	HBSS
鸡	小鸡胚胎	Retinal	Trypsin: 0.6%	DMEM	Seigel, G.: The Golden Age of Retinal Cell Culture, Mol Vis 5, 4, 1999
鸡	(包括大鼠)	Retinal	Trypsin: 0.6%	DMEM
鸡	小鸡, 胚胎, 10-14 天	Flat, retina	Trypsin: 0.1%	Tyrode's solution, CMF	Moyer, M., Bullrich, F., and Sheffield, J.: Emergence of Flat Cells From Glia in Stationary Cultures of Embryonic Chick Neural Retina, In Vitro Cell Dev Biol 26, 1073, 1990
鱼类	鱼, 斑马鱼	Retinal	Papain: 10 u/ml	L-15	Diekmann, H., Kalbhen, P. and Fischer, D.: Characterization of Optic Nerve Regeneration using Transgenic Zebrafish., Front Cell Neurosci 9, 118, 2015
人类	人类	Corneal stromal stem	Neutral Protease: 1.2 u/ml	DMEM	Du, Y., Roh, D., Funderburgh, M., Mann, M., Marra, K., Rubin, J., Li, X. and Funderburgh, J.: Adipose-Derived Stem Cells Differentiate to Keratocytes In Vitro.,Mol Vis 16, 2680, 2010
人类	人类	Corneal stromal stem	Collagenase: 0.1%	DMEM
人类	人类, 18-68 岁	Corneal endothelial	Collagenase: 0.2%	DMEM/F12	Li Wei, Sabater Alfonso L, Chen Ying-Ting, Hayashida Yasutaka, Chen Szu-Yu, He Hua, Tseng Scheffer C G: A novel method of isolation, preservation, and expansion of human corneal endothelial cells, Inv Ophthal Visual Sci 48, 614-20, 2007
人类	人类, 18-68 岁	Corneal endothelial	Neutral Protease: 1.0%	DMEM/F12
人类	人类	Corneal limbal epithelial sheet	Neutral Protease: 5%	see reference	Espana EM, Romano AC, Kawakita T, Di Pascuale M, Smiddy R, and Tseng SC:Novel enzymatic isolation of an entire viable human limbal epithelial sheet, Inv Ophthal Visual Sci 44(10), 4275, 2003
人类	人类, 5-65 岁	Retinal pigment epithelial (RPE)	Trypsin: 0.25%	HBSS	Von Recum, H., Okano, T., Kim, S, and Bernstein, P.: Maintenance of Retinoid Metabolism in Human Retinal Pigment Epithelium Cell Culture, Exp Eye Res 69, 97, 1999
猴	猕猴和狒狒	Retinal	Papain: 20-40 u/ml	Ames' solution	Han ,Y., Jacoby, R. and Wu, S.: Morphological and Electrophysiological Properties of Dissociated Primate Retinal Cells., Brain Res 875, 175, 2000
猴	猕猴和狒狒	Retinal	Deoxyribonuclease I: 400 u/ml	Ames' solution
猴	猴, 猕猴, 青少年	Conjunctival lymphocytes	Collagenase Type 1: 0.02%	RPMI 1640	Whittum-Hudson JA, Taylor HR: Antichlamydial specificity of conjunctival lymphocytes during experimental ocular infection, Infect Immun 57, 2977, 1989
小鼠	小鼠, 成年的	Retinal photoreceptor	PDS kit: with modifications	EBSS	Feodorova, Y., Koch, M., Bultman, S., Michalakis, S. and Solovei, I.: Quick and Reliable Method for Retina Dissociation and Separation of Rod Photoreceptor Perikarya from Adult Mice., MethodsX 2, 39, 2015
小鼠	小鼠, 4-8 天	Photoreceptors	PDS kit: per instructions	Neurobasal	Balmer, J., Zulliger, R., Roberti, S. and Enzmann, V.: Retinal Cell Death Caused by Sodium Iodate Involves Multiple Caspase-Dependent and Caspase-Independent Cell-Death Pathways., Int J Mol Sci 16, 15086-103, 2015
小鼠	小鼠, 胚胎和 P0蛋白	Retinal	Papain: 8 u/ml	DPBS	Dvoriantchikova, G., Perea-Martinez, I., Pappas, S., Barry, A., Danek, D., Dvoriantchikova, X., Pelaez, D. and Ivanov, D.: Molecular Characterization of Notch1 Positive Progenitor Cells in the Developing Retina., PLoS ONE 10, e0131054, 2015
小鼠	小鼠, 胚胎和 P0蛋白	Retinal	Deoxyribonuclease I: 124 u/ml	DPBS
小鼠	小鼠	Vitreous cells	Collagenase: 0.1%	DMEM	Iqbal, N., Xu, L., Devitt, C. and Skapek, S.: Isolation and Characterization of Mammalian Cells Expressing the Arf Promoter During Eye Development.,Biotechniques 56, 239-49, 2014
小鼠	小鼠	Vitreous cells	Hyaluronidase: 0.03%	DMEM
小鼠	小鼠, 6 周	Retinal	PDS kit: per instructions	DMEM	Singh, M., Issa, P, Butler, R., Martin, C., Lipinski, D., Sekaran, S., Barnard, A. and Maclaren, R.: Reversal of End-Stage Retinal Degeneration and Restoration of Visual Bunction by Photoreceptor Transplantation., Proc Natl Acad Sci U S A , , 2013
小鼠	小鼠	Retinal ganglion	CLSPA: 240 u/ml	Ames	Schmidt, T. and Kofuji, P.: An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells., J Vis Exp 47, 2367, 2011
小鼠	小鼠	Retinal ganglion	Hyaluronidase: 0.2%	Ames
小鼠	小鼠, 成年的	Cilary epithelial	PDS kit: per instructions	EBSS	Gualdoni, S., Baron, M., Lakowski, J., Decembrini, S., Pearson, R., Ali, R. and Sowden, J.: Isolation and Culture of Adult Ciliary Epithelial Cells, Previously Identified as Retinal Stem Cells, and Retinal Progenitor Cells., Curr Protoc Stem Cell Biol Chapter 1, Unit 1H.4, 2011
小鼠	小鼠, 出生后一天的	Retinal progenitor	Collagenase: 0.1%	HBSS	Jiang, C. Klassen, H., Zhang, X. and Young, M.: Laser Injury Promotes Migration and Integration of Retinal Progenitor Cells into Host Retina., Mol Vis 16, 983, 2010
小鼠	小鼠	Retinal	Collagenase: 0.1%	RPMI	Amadi-Obi, A., Yu, C., Liu, X., Mahdi, R., Clarke, G., Nussenblatt, R, Gery, I., Lee, Y. and Egwuagu, C.: TH17 Cells Contribute to Uveitis and Scleritis and are Expanded by IL-2 and Inhibited by IL-27/STAT1., Nat Med 13, 711, 2007
小鼠	小鼠	Retinal	Deoxyribonuclease I: 0.001%	RPMI
小鼠	小鼠, 成年的	Retinal	Papain: 50 u/ml	DMEM/F-12	Jadhav AP, Cho SH and Cepko CL: Notch activity permits retinal cells to progress through multiple progenitor states and acquire a stem cell property, Proc Natl Acad Sci U S A 103, 18998, 2006
小鼠	小鼠, 2-5 个月	Mouse retinal and bipolar	Papain: 20 u/ml	HBSS	Maxeiner Stephan, Dedek Karin, Janssen-Bienhold Ulrike, Ammermuller Josef, Brune Hendrik, Kirsch Taryn, Pieper Mario, Degen Joachim, Kruger Olaf, Willecke Klaus, Weiler Reto: Deletion of connexin45 in mouse retinal neurons disrupts the rod/cone signaling pathway between AII amacrine and ON cone bipolar cells and leads to impaired visual transmission, J Neurosci 25, 566-76, 2005
小鼠	小鼠, 2-5 个月	Mouse retinal and bipolar	Deoxyribonuclease I: 200 u/ml	HBSS
小鼠	小鼠, 4 周	Retinal endothelial cells	Collagenase Type 1: 0.1%	DMEM	Su X, Sorenson CM, and Sheibani N.: Isolation and characterization of murine retinal endothelial cells, Mol Vis 9, 171, 2003
猪	猪, 5-60 公斤	Retinal pigment epithelial cells	Collagenase: 2%	DMEM	Wiencke, A., Kiilgaard, J., Nicolini, J., Bundgaard, M., Ropke, C., and La Cour, M.: Growth of Cultured Porcine Retinal Pigment Epithelial Cells, Acta Opthalmol Scand 81(2), 170, 2003
兔	兔, New Zealand, Adult	Corneal Endothelial	Collagenase Type 2: 0.2%	HBSS	Lai, J., Cheng, H. and Ma, D.: Investigation of Overrun-Processed Porous Hyaluronic Acid Carriers in Corneal Endothelial Tissue Engineering, PLoS ONE 10, 1371, 2015
兔	兔	Corneal keratocytes	Hyaluronidase: 0.05%	DMEM	Hao, M., Flynn, K., Nien-Shy, C., Jester, B., Winkler, M., Brown, D., La Schiazza, O., Bille, J., Jester, J.: In Vivo Non Linear Optical (NLO) Imaging in Live Rabbit Eyes Using the Heidelbert Two-Photon Laser Opthalmoscope, Exp Eye Res 91, 308, 2010
兔	兔	Corneal keratocytes	Collagenase: 0.2%	DMEM
兔	兔, 新西兰	Corneal keratocytes	Trypsin: 0.25%	PBS	Stramer Brian M, Kwok Michael G K, Farthing-Nayak Pamela J, Jung Jae-Chang, Fini M Elizabeth, Nayak Ramesh C: Monoclonal antibody (3G5)-defined ganglioside: cell surface marker of corneal keratocytes, Inv Ophthal Visual Sci 45, 807-12, 2004
兔	兔, 新西兰	Corneal keratocytes	Collagenase: 0.5%	PBS
兔	兔, 成年的	Retinal neurons	Papain: 26 u/ml	DMEM	Brockway LM, Zhou Z-H, Bubien JK, Jovov B, Benos DJ, Keyser KT: Rabbit retinal neurons and glia express a variety of ENaC/DEG subunits, Am J Physiol Cell Physiol283, C126-34, 2002
兔	兔, 新西兰, 成年的, 雄性, 白化变种,	Epithelial	Trypsin: 0.25%	HBSS/DMEM	Johnson-Muller, B., and Gross, J.: Regulation of Corneal Collagenase Production: Epithelial-Stromal Cell Interactions, Proc Natl Acad Sci U S A 75 (9), 4417, 1978
兔	2 公斤	Epithelial	Trypsin: 0.25%	HBSS/DMEM
大鼠	SD大鼠,6-8 周	Retinal	PDS kit: per instructions	EBSS	Vigneswara, V., Esmaeili, M., Deer, L., Berry, M., Logan, A. and Ahmed, Z.: Eye Drop Delivery of Pigment Epithelium-Derived Factor-34 Promotes Retinal Ganglion Cell Neuroprotection and Axon Regeneration., Mol Cell Neurosci 68, 212-21, 2015
大鼠	大鼠, SD	Retinal ganglion	PDS kit: with modifications	Neurobasal	Tan, C., Andrews, M., Kwok, J., Heintz, T., Gumy, L., Fassler, R. and Fawcett, J.:Kindlin-1 Enhances Axon Growth on Inhibitory Chondroitin Sulfate Proteoglycans and Promotes Sensory Axon Regeneration., J Neurosci 32, 7325, 2012
大鼠	SD大鼠, 180-220克	Retinal ganglion	Papain: 0.2%	Neurobasal	Ma, J., Yu, W., Wang, Y., Cao, G., Cai, S., Chen, X., Yan, N., Yuan, Y., Zeng, H., Fleenor, D., Liu, X. and Pang, I.: Neuroprotective Effects of C-type Natriuretic Peptide on Rat Retinal Ganglion Cells., Inv Ophthal Visual Sci 51, 3544-53, 2010
大鼠	Fishe大鼠, 成年的	Retinal	PDS kit: per instructions	MEM	Suzuki, T., Mandai, M., Akimoto, M., Yoshimura, N. and Takahashi, M.: The Simultaneous Treatment of MMP-2 Stimulants in Retinal Transplantation Enhances Grafted Cell Migration into the Host Retina., Stem Cells 24, 2406, 2006
大鼠	大鼠, 暗条纹, 3-4 周	Ciliary-derived eye	PDS kit: with modifications	DMEM/F12	Akagi T, Mandai M, Ooto S, Hirami Y, Osakada F, Kageyama R, Yoshimura N, Takahashi M.: Otx2 homeobox gene induces photoreceptor-specific phenotypes in cells derived from adult iris and ciliary tissue, Inv Ophthal Visual Sci 45, 4570, 2004
大鼠	大鼠, 胚胎 17 天	Retinal	Trypsin: 0.6%	DMEM	Seigel, G.: The Golden Age of Retinal Cell Culture, Mol Vis 5, 4, 1999
大鼠	大鼠, SD	Retina	Papain: 120 U	HBSS, PBS	Jing, S., Wen, D., Yu, Y., Holst, P., Luo, Y., Fang, M., Tamir, R., Antonio, L., Hu, Z., et al.: GDNF-Induced Activation of the Ret Protein Tyrosine Kinase is Mediated by GDNFR-a, a Novel Receptor for GDNF, Cell 85, 1113, 1996
大鼠	大鼠, 4-14 week	Retinal pigment epithelial cells	Collagenase Type 1: 65 u/ml	CF Hanks with EDTA	Wang N, Koutz CA, and Anderson RE.: A method for the isolation of retinal pigment epithelial cells from adult rats, Inv Ophthal Visual Sci 34(1), 101, 1993
大鼠	大鼠, 4-14 week	Retinal pigment epithelial cells	Hyaluronidase: 220 u/ml	CF Hanks with EDTA
大鼠	大鼠, 6-8 天	Retinal pigment epithelial	Neutral Protease: 2%	DMEM	Chang CW, Roque RS, Defoe DM, and Caldwell RB.: An improved method for isolation and culture of pigment epithelial cells from rat retina, Curr Eye Res 10(11), 1081, 1991
大鼠	大鼠, 长伊万斯, 1-15 天	Neurons, visual cortex	Papain: 20 u/ml	BSS	Huettner, J., and Baughman, R.: Primary Culture of Identified Neurons From the Visual Cortex of Postnatal Rats, J Neurosci 6, 3044, 1986
大鼠	大鼠, 长伊万斯, 1-15 天	Neurons, visual cortex	Papain: 20 u/ml	(see reference)
大鼠	大鼠, SD, 小狗	Retina	Trypsin: 0.25%	Ham's F-12	Sarthy PV, Curtis BM, and Catterall WA.: Retrograde Labeling, Enrichment, and Characerization of Retinal Ganglion Cells from the Neonatal Rat, J Neurosci 3 (12), 2532, 1983
蝾螈	蝾螈	Neurons	Papain: 14 u/ml	Ringers	Clarke, R, Wang, J. and Townes-Anderson, E.: Using Laser Tweezers For Manipulating Isolated Neurons In Vitro., J Vis Exp 19, 911, 2008
蝾螈	蝾螈, 18-25 厘米	Retina	Papain: 14 u/ml	Saline	Townes-Anderson, E., MacLeish, P., and Raviola, E.: Rod Cells Dissociated from Mature Salamander Retina: Ultrastruture and Uptake of Horseradish Peroxidase, J Cell Biol 100, 175, 1985
蝾螈	蝾螈 (A. tigrinum)	Photoreceptors, retina	Papain: 0.05%	(see reference)	Bader, C., MacLeish, P., and Schwartz, E.: Responses to Light of Solitary Rod Photoreceptors Isolated From Tiger Salamander Retina, Proc Natl Acad Sci U S A 75, 3507, 1978
龟	龟(红耳龟)	Retinal	Papain: 0.1% (13.5 u/mg)	Kreb's Ringer	Lam, D.: Biosynthesis of Acetylcholine in Turtle Photoreceptors, Proc Natl Acad Sci U S A 69, 1987, 1972

我们公司zui大优势是强大的采购，

1：基本什么都能进口，血清，抗体，耗材，还有部分限制进口的，

2：货品全，现经营过700多个品牌，基本所有生物试剂耗材都可以进口，特别是冷偏的产品那就更有优势，

3：提供加急服务，一般1-2周到货，超过时限加急费全免

4：价格公道，绝大部分价格有优势，当然不能保证100%产品都是,因为意味着没有服务.

5：良好的信誉，大部分客户我们提供货到付款服务，客户包括清华，北大交大复旦，中山等100多所大学，ROCHE，阿斯利康，国药，fisher等500多家公司

6：我们还是Santa,Advanced Biotechnologies Inc, Worthington Biochemical ,bangs,BBInternational,crystalchem,dianova,FD Neurotechnologies,Inc. Dako,Inc, GePromegaridege, Glycotope Biotechnology GmbH; iduron,Innovative Research of America, Ludger, neuroprobe,Worthington Biochemical , Polysciences,prospecbi, QA-BIO,quickzyme,FULLERLABS,INC,sterlitech；sysy,TriLink BioTechnologies,Inc；worthington-biochem,zyagen等几十家国外公司授权代理。
7：我们还是invitrogen,qiagen,Worthington Biochemical am,sigma;Promega,roche,merck, rnd,BD, GE,pierce,BioLegend等*批发，欢迎合作。

上一条：心脏组织消化手册
下一条：上皮细胞组织消化手册