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眼組織消化手冊(cè)

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Worthington組織消化指南

引言

組織消化酶主要用于組織培養(yǎng)和細(xì)胞生物學(xué)研究的原代細(xì)胞的分離和收集。盡管這些消化酶已經(jīng)普遍應(yīng)用多年,但是對(duì)其組織消化和細(xì)胞收集的作用機(jī)制卻沒(méi)有全面的理解。  由此導(dǎo)致的結(jié)果便是選擇一種消化方法大多是隨機(jī)選擇或基于過(guò)去的經(jīng)驗(yàn),而不是在*理解消化原理的基礎(chǔ)上選擇的消化方案,以及如何根據(jù)具體實(shí)驗(yàn)?zāi)康男拚桨敢垣@得更好的實(shí)驗(yàn)結(jié)果。

細(xì)胞分離程序的目的是zui大程度的獲得功能活性的單個(gè)細(xì)胞,而影響結(jié)果的原因有很多,包括(不限于)以下:

1、組織類(lèi)型

2、物種來(lái)源

3、動(dòng)物年齡

4、遺傳改造情況(如knockouts

5、所用的消化培養(yǎng)液

6、所用的酶

7、消化酶中的雜質(zhì)

8、所用消化酶的工作濃度

9、溫度

10、孵育時(shí)間

通常前四個(gè)原因沒(méi)辦法選擇,而剩下的原因一般由經(jīng)驗(yàn)決定以獲取zui適實(shí)驗(yàn)結(jié)果。

通過(guò)查閱文獻(xiàn)來(lái)選擇*組織消化酶和zui適消化條件的研究人員通常被互相矛盾的實(shí)驗(yàn)數(shù)據(jù)搞得一頭霧水。這些巨大差異來(lái)源于組織胞外基質(zhì)的復(fù)雜而動(dòng)態(tài)的特性,以及過(guò)去常用的粗制、未提純的組織分離酶。另外,組織胞外基質(zhì)由各種蛋白、糖蛋白、脂類(lèi)和糖脂組成,這些成分的組成比例在不同物種、不同組織、甚至不同發(fā)育(年齡)之間存在明顯的差異,這些因素都極大的影響了消化方案的制定。目前通常使用的粗制酶,如鏈霉蛋白酶、NF1:250、以及含有幾種其他不同濃度蛋白酶的膠原酶、以及各種多糖酶、核酶和脂酶。

本指南總結(jié)了目前常用組織消化酶的zui適消化和細(xì)胞收集知識(shí)和方法,描述了標(biāo)準(zhǔn)的實(shí)驗(yàn)步驟, 便于制定一個(gè)*的細(xì)胞分離方案,同時(shí)列舉了一些相關(guān)的參考文獻(xiàn)。

下表列出多種組織的適宜消化酶(含參考文獻(xiàn),以組織類(lèi)型和物種分類(lèi),詳見(jiàn)鏈接)

脂肪組織

腎上腺組織

骨組織

腦組織

軟骨組織

結(jié)腸組織

內(nèi)皮組織

上皮組織

心臟

淋巴結(jié)

乳腺

其它組織

其他組織

肌組織

神經(jīng)組織

胰腺

腮腺

垂體

前列腺

生殖

動(dòng)物鱗片

皮膚

脾臟

干細(xì)胞

胸腺

甲狀腺/甲狀旁腺

扁桃體

腫瘤

 

 

 

注:使用Worthington公司消化酶的文獻(xiàn)不限于上表鏈接中所列,一般而言,Worthington公司的組織消化酶可用于大多組織類(lèi)型的消化。

 

眼組織消化手冊(cè)

 

物種詳細(xì)來(lái)源細(xì)胞名稱(chēng)消化用酶培養(yǎng)基參考文獻(xiàn)
PericyteCollagenase: 0.2%DMEMBryan, B. and D'Amore, P.: Pericyte Isolation and USe is Endothelial/Pericyte Coculture Models, Meth Enzymol 443, 315, 2008
Microvascular endothelialCollagenase/Dispase: 0.1%MEMBowman, P., Betz, A., and Goldstein, G.: Primary Culture of Microvascular Endothelial Cells From Bovine Retina, In Vitro 18 (7), 626, 1982
小雞, 胚胎, 6 天Retinal cellsTrypsin: 0.005%DMEM/F12Jacob 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
Deoxyribonuclease I: 0.005%
小雞 胚胎Corneal epitheliaCollagenase: 0.08%HBSSReenstra 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
Trypsin: 0.08%
小雞 胚胎RetinalTrypsin: 0.6%DMEMSeigel, G.: The Golden Age of Retinal Cell Culture, Mol Vis 5, 4, 1999
(包括大鼠)
小雞, 胚胎, 10-14 天Flat, retinaTrypsin: 0.1%Tyrode's solution, CMFMoyer, 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
魚(yú)類(lèi)魚(yú), 斑馬魚(yú)RetinalPapain: 10 u/mlL-15Diekmann, H., Kalbhen, P. and Fischer, D.: Characterization of Optic Nerve Regeneration using Transgenic Zebrafish., Front Cell Neurosci 9, 118, 2015
人類(lèi)人類(lèi)Corneal stromal stemNeutral Protease: 1.2 u/mlDMEMDu, 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
Collagenase: 0.1%
人類(lèi)人類(lèi), 18-68 歲Corneal endothelialCollagenase: 0.2%DMEM/F12Li 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
Neutral Protease: 1.0%
人類(lèi)人類(lèi)Corneal limbal epithelial sheetNeutral Protease: 5%see referenceEspana 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
人類(lèi)人類(lèi), 5-65 歲Retinal pigment epithelial (RPE)Trypsin: 0.25%HBSSVon 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
獼猴和狒狒RetinalPapain: 20-40 u/mlAmes' solutionHan ,Y., Jacoby, R. and Wu, S.: Morphological and Electrophysiological Properties of Dissociated Primate Retinal Cells., Brain Res 875, 175, 2000
Deoxyribonuclease I: 400 u/ml
猴, 獼猴, 青少年Conjunctival lymphocytesCollagenase Type 1: 0.02%RPMI 1640Whittum-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 photoreceptorPDS kit: with modificationsEBSSFeodorova, 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PhotoreceptorsPDS kit: per instructionsNeurobasalBalmer, 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RetinalPapain: 8 u/mlDPBSDvoriantchikova, 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
Deoxyribonuclease I: 124 u/ml
小鼠小鼠Vitreous cellsCollagenase: 0.1%DMEMIqbal, 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
Hyaluronidase: 0.03%
小鼠小鼠, 6 周RetinalPDS kit: per instructionsDMEMSingh, 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 ganglionCLSPA: 240 u/mlAmesSchmidt, T. and Kofuji, P.: An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells., J Vis Exp 47, 2367, 2011
Hyaluronidase: 0.2%
小鼠小鼠, 成年的Cilary epithelialPDS kit: per instructionsEBSSGualdoni, 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 progenitorCollagenase: 0.1%HBSSJiang, 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RetinalCollagenase: 0.1%RPMIAmadi-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
Deoxyribonuclease I: 0.001%
小鼠小鼠, 成年的RetinalPapain: 50 u/mlDMEM/F-12Jadhav 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 個(gè)月Mouse retinal and bipolarPapain: 20 u/mlHBSSMaxeiner 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
Deoxyribonuclease I: 200 u/ml
小鼠小鼠, 4 周Retinal endothelial cellsCollagenase Type 1: 0.1%DMEMSu X, Sorenson CM, and Sheibani N.: Isolation and characterization of murine retinal endothelial cells, Mol Vis 9, 171, 2003
豬, 5-60 公斤Retinal pigment epithelial cellsCollagenase: 2%DMEMWiencke, 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, New Zealand, AdultCorneal EndothelialCollagenase Type 2: 0.2%HBSSLai, J., Cheng, H. and Ma, D.: Investigation of Overrun-Processed Porous Hyaluronic Acid Carriers in Corneal Endothelial Tissue Engineering, PLoS ONE 10, 1371, 2015
Corneal keratocytesHyaluronidase: 0.05%DMEMHao, 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
Collagenase: 0.2%
兔, 新西蘭Corneal keratocytesTrypsin: 0.25%PBSStramer 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
Collagenase: 0.5%
兔, 成年的Retinal neuronsPapain: 26 u/mlDMEMBrockway 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, 新西蘭, 成年的, 雄性, 白化變種, EpithelialTrypsin: 0.25%HBSS/DMEMJohnson-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
2 公斤
大鼠 SD大鼠,6-8 周RetinalPDS kit: per instructionsEBSSVigneswara, 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, SDRetinal ganglionPDS kit: with modificationsNeurobasalTan, 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 ganglionPapain: 0.2%NeurobasalMa, 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RetinalPDS kit: per instructionsMEMSuzuki, 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, 暗條紋, 3-4 周Ciliary-derived eyePDS kit: with modificationsDMEM/F12Akagi 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RetinalTrypsin: 0.6%DMEMSeigel, G.: The Golden Age of Retinal Cell Culture, Mol Vis 5, 4, 1999
大鼠大鼠, SDRetinaPapain: 120 UHBSS, PBSJing, 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 weekRetinal pigment epithelial cellsCollagenase Type 1: 65 u/mlCF Hanks with EDTAWang 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
Hyaluronidase: 220 u/ml
大鼠大鼠, 6-8 天Retinal pigment epithelialNeutral Protease: 2%DMEMChang 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, 長(zhǎng)伊萬(wàn)斯, 1-15 天Neurons, visual cortexPapain: 20 u/mlBSSHuettner, J., and Baughman, R.: Primary Culture of Identified Neurons From the Visual Cortex of Postnatal Rats, J Neurosci 6, 3044, 1986
(see reference)
大鼠大鼠, SD, 小狗RetinaTrypsin: 0.25%Ham's F-12Sarthy 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
蠑螈蠑螈NeuronsPapain: 14 u/mlRingersClarke, R, Wang, J. and Townes-Anderson, E.: Using Laser Tweezers For Manipulating Isolated Neurons In Vitro., J Vis Exp 19, 911, 2008
蠑螈蠑螈, 18-25 厘米RetinaPapain: 14 u/mlSalineTownes-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
蠑螈蠑螈 (A. tigrinum)Photoreceptors, retinaPapain: 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
龜(紅耳龜)RetinalPapain: 0.1% (13.5 u/mg)Kreb's RingerLam, D.: Biosynthesis of Acetylcholine in Turtle Photoreceptors, Proc Natl Acad Sci U S A 69, 1987, 1972

 

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6:我們還是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等幾十家國(guó)外公司授權(quán)代理。
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