腎組織消化手冊

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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，以便于制定一個*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
眼	心臟	腸	腎
肝	肺	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。

腎組織消化手冊

物種	詳細來源	細胞名稱	消化用酶	培養基	參考文獻
鳥類	鳥類, 麻雀	Tubule segments	Collagenase Type 2: 0.1%	HBSS	Goldstein D., Reddy V., and Plaga K.: Second Messenger Production in Avian Medullary Nephron Segments in Response to Peptide Hormones, Am J Physiol 276, R847, 1999
鳥類	鳥類, 雞	Tubule and glomeruli	Collagenase Type 1: 0.1%	RPMI 1640	Reich, C. and Bonar, R.: Separation of Avian Kidney Tubules and Glomeruli for In Vitro Culture, Meth Cell Sci 7, 97, 1982
牛	牛	Papillary duct	Hyaluronidase: 0.2%	Keri's buffer	Husted, R., Hayashi, M., and Stokes, J.: Characteristics of Papillary Collecting Duct Cells in Primary Culture, Am J Physiol 255, F1160, 1988
牛	牛	Papillary duct	Hyaluronidase: 0.2%	HEPES buffered saline
犬	犬	Renal adipose derived cells	Collagenase Type 1: 0.3%	DMEM	Basu, J., Genheimer, C., Sangha, N., Quinlan, S., Guthrie, K., Kelley, R., Ilagan, R., Jain, D., Bertram, T. and Ludlow, J.: Organ Specific Regenerative Markers in Peri-Organ Adipose: Kidney., Lipids Health Dis 10, 171, 2011
犬	狗, 1-5 個月	Proximal tubules	Collagenase Type 1: 0.15%	Krebs Ringer bicarbonate buffer	States, B., Reynolds, R., Lee, J., and Segal, S.: Cystine Uptake By Cultured Cells Originating From Dog Proximal Tubule Segments, In Vitro Cell Dev Biol 26, 105, 1990
犬	狗, 雜種狗, 20 公斤	Proximal tubular	Deoxyribonuclease I: 0.0125%	(see reference)	Yau, C., Rao, L., and Silverman, M.: Sugar Uptake Into a Primary Culture of Dog Kidney Proximal Tubular Cells, Can J Physiol Pharmacol 63, 417, 1985
魚	美洲擬鰈 (鈍吻黃蓋鰈)	Renal tubule	Trypsin: 0.2%	CMF solution	Dickman, K. and Renfro, J.: Primary Culture of Flounder Renal Tubule Cells:Transepithelial Transport, Am J Physiol 251, 424, 1986
魚	200 - 500 克	Renal tubule	Trypsin: 0.2%	(see reference)
豚鼠	豚鼠, 雄性, 100 克	Single cells	Trypsin: 0.25%	CF salt solution	Phillips, H.: Dissociation of Single Cells from Lung or Kidney Tissue with Elastase, In Vitro 8, 101, 1972
倉鼠	倉鼠	Papillary duct	Hyaluronidase: 0.2%	Keri's buffer	Husted, R., Hayashi, M., and Stokes, J.: Characteristics of Papillary Collecting Duct Cells in Primary Culture, Am J Physiol 255, F1160, 1988
倉鼠	(包括SD大鼠, Wistar大鼠, 150-225 克, 兔, 牛)	Papillary duct	Hyaluronidase: 0.2%	HEPES buffered saline
人類	人類	Renal adipose derived cells	Collagenase Type 1: 0.3%	DMEM	Basu, J., Genheimer, C., Sangha, N., Quinlan, S., Guthrie, K., Kelley, R., Ilagan, R., Jain, D., Bertram, T. and Ludlow, J.: Organ Specific Regenerative Markers in Peri-Organ Adipose: Kidney., Lipids Health Dis 10, 171, 2011
人類	人類, 成年人	Renal tumor cells and proximal tubular epithelial	Collagenase Type 2: 0.1%	DMEM/F12	Valente, M., Henrique, R., Costa, V., Jeronimo, C., Carvalho, F., Bastos, M., Guedes de Pinho, P. and Carvalho, M.: A Rapid and Simple Procedure for the Establishment of Human Normal and Cancer Renal Primary Cell Cultures from Surgical Specimens., PLoS ONE 6, e19337, 2011
人類	人類	Renal cells	Collagenase Type 4: 300 u/ml	HBSS	Presnell, S., Bruce, A., Wallace, S., Choudhury, S., Genheimer, C., Cox, B., Guthrie, K., Werdin, E., Tatsumi-Ficht, P., Ilagan, R., Kelley, R., Rivera, E., Ludlow, J., Wagner, B., Jayo, M. and Bertram, T.: Isolation, Characterization, and Expansion Methods for Defined Primary Renal Cell Populations from Rodent, Canine, and Human Normal and Diseased Kidneys., Tiss Eng 17, 261, 2011
人類	人類	Renal cells	Neutral Protease: see reference	HBSS
人類	人類, 成年人	Renal proximal tubule and cortical fibroblasts	Collagenase Type 2: 383 u/ml	DMEM/F-12	Johnson, D., Saunders, H., Johnson, F., Huq, S., Field, M., and Pollock, C.: Cyclosporin exerts a direct fibrogenic effect on human tubulointerstitial cells: roles of insulin-like growth factor I, transforming growth factor beta1, and plaet-derived growth factor, J Pharmacol Exp Ther 289, 535-42, 1999
人類	人類	Renal cortex	Trypsin: 0.1%	Tissue Culture Grade Water	McAteer, J, Kempson, S., and Evan, A: Culture of Human Renal Cortex Epithelial Cells, J Tiss Cul Meth 13, 143, 1991
人類	人類	Papillary duct	Collagenase: 400 u/ml	Eagle's MEM-HEPES buffer w/L-glutamine	Trifillis, A. and Kahng, M.: Characterization of an In Vitro System of Human Renal Papillary Collecting Duct Cells, In Vitro Cell Dev Biol 26, 441, 1990
人類	人類	Mesangial	Trypsin: 0.25%	DMEM/Ham's F-12	Heieren, M., van der Woude, F., and Balfour Jr., H.: Cytomegalovirus Replicates Efficiently in Human Kidney Mesangial Cells, Proc Natl Acad Sci U S A 85, 1642, 1988
人類	人類, 成年人	Tubular	Collagenase: 250 u/ml	PBS	Yang, A., Gould-Kostka, J., and Oberley, T.: In Vitro Growth and Differentiation of Human Kidney Tubular Cells on a Basement Membrane Substrate, In Vitro Cell Dev Biol 23 (1), 34, 1987
人類	人類, 成年人, 14-66 歲	Tubular	Collagenase: 100 u/ml	Joklik's MEM	Trifillis, A., Regec, A., and Trump, B.: Isolation, Culture, and Charaterization of Human Renal Tubular Cells, J Urol 133, 324, 1985
人類	人類	Malignant	Papain: 0.009%	Sacks solution	Hemstreet, G., Enoch, P., and Pretlow, T.: Tissue Disaggregation of Human Renal Cell Carcinoma with Further Isopyknic and Isokinetic Gradient Purification, Cancer Res 40, 1043, 1980
人類	人類	Stromal	Papain: 0.009%	Sacks solution
人類	人類, 24 初生和夭折的	Renal	Trypsin: 0.25%	See reference	De Oca, H., and Malinin, T.: Dispersion and C*tion of Renal Cells After Short-Term Storage of Kidneys, J Clin Microbiol 2, 243, 1975
人類	(包括兔)	Renal	Trypsin: 0.25%	See reference
猴	猴	Kidney	Trypsin: 0.01% - 0.00001%	Eagle's MEM	Melnick, J., and Wallis, C.: Problems Related to the Use of Serum and Trypsin in the Growth of Monkey Kidney Cells, Dev Biol 37, 77, 1976
小鼠	小鼠	Collecting duct epithelial	Collagenase Type 2: 0.1%	DMEM	DiRocco, D., Kobayashi, A., Taketo, M., McMahon, A. and Humphreys, B.: Wnt4/尾-catenin Signaling in Medullary Kidney Myofibroblasts., J Am Soc Nephrol 24, 1399, 2013
小鼠	小鼠	Glomeruli	Collagenase Type 4: 0.1%	RPMI 1640	Kabgani, N., Grigoleit, T., Schulte, K., Sechi, A., Sauer-Lehnen, S., Tag, C., Boor, P., Kuppe, C., Warsow, G., Schordan, S., Mostertz, J., Chilukoti, R., Homuth, G., Endlich, N., Tacke, F. and Weiskirchen, R.: Primary Cultures of Glomerular Parietal Epithelial Cells or Podocytes with Proven Origin., PLoS ONE 7, e34907, 2012
小鼠	小鼠, 10-14 week	Proximal tubule	Collagenase Type 1: 0.1%	See reference	Park, J., Pasupulati, R., Feldkamp, T., Roeser, N. and Weinberg, J.: Cyclophilin D and the Mitochondrial Permeability Transition in Kidney Proximal Tubules after Hypoxic and Ischemic Injury., Am J Physiol/Renal 301, F134, 2011
小鼠	小鼠, 胚胎	Kidney	Collagenase Type 1: 0.25%	HBSS	Brown, A., Blank, U., Adams, D., Karolak, M., Fetting, J., Hill, B. and Oxburgh, L.:Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney., J Vis Exp 50, e2555, 2011
			Pancreatin: 1.0%
			Deoxyribonuclease I: 1 u/ml
小鼠	小鼠,成年的	Kidney Sphere-Derived	Collagenase Type 4: 0.1%	Iscove's Modified Dulbecco's	Huang, Y., Johnston, P., Zhang, B., Zakari, A., Chowdhry, T., Smith, R., Marban, E., Rabb, H. and Womer, K.: Kidney-Derived Stromal Cells Modulate Dendritic and T Cell Responses., J Am Soc Nephrol 20, 831-41, 2009
小鼠	小鼠, 雄性, 8-16 周	Renal tubular epithelial	Collagenase: 200 u/ml	HBSS	Breggia, A. and Himmelfarb, J.: Primary Mouse Renal Tubular Epithelial Cells have Variable Injury Tolerance to Ischemic and Chemical Mediators of Oxidative Stress., Oxid Med Cell Longev 1, 33, 2008
小鼠	小鼠, 雄性, 8-16 周	Renal tubular epithelial	Soybean Trypsin Inhibitor: see reference	HBSS
小鼠	小鼠	Proximal tubule	Collagenase Type 2: 0.013%	see reference	Wright, J., Morales, M., Sousa-Menzes, J., Ornellas, D., Sipes, J., Cui, Y., Cui, I., Hulamm, P., Cebotaru, V., Cebotaru, L., Guggino, W. and Guggino, S.: Transcriptional Adaptation to Clcn5 Knockout in Proximal Tubules of Mouse Kidney., Physiol Genomics 33, 341, 2008
小鼠	小鼠, 4 周	Endothelial kidney	Collagenase Type 1: 0.1%	DMEM	Kondo, S., Scheef, E., Sheibani, N. and Sorenson, C.: PECAM-1 Isoform-Specific Regulation of Kidney Endothelial Cell Migration and Capillary Morphogenesis., Am J Physiol Cell Physiol 292, C2070, 2007
小鼠	小鼠, 成年的	Cortex, proximal tubule	Collagenase: 0.15%	DMEM	Syal Ashu, Schiavi Susan, Chakravarty Sumana, Dwarakanath Vangipuram, Quigley Raymond, Baum Michel: Fibroblast growth factor-23 increases mouse PGE2 production in vivo and in vitro, Am J Physiol/Renal 290, F450-5, 2006
小鼠	小鼠	Kidney Mesenchymal	Collagenase: 250 u/ml	DMEM/F12	Plotkin, M. and Goligorsky, M.: Mesenchymal Cells from Adult Kidney Support Angiogenesis and Differentiate into Multiple Interstitial Cell Types including Erythropoietin-Producing Fibroblasts., Am J Physiol Renal Physiol 291, F902-12, 2006
小鼠	小鼠	Kidney Mesenchymal	Neutral Protease: 33.3 u/ml	DMEM/F12
小鼠	小鼠, 10-12 周	Kidney	Collagenase Type 4: 0.075%	DMEM	Dekel, B., Zangi, L., Shezen, E., Reich-Zeliger, S., Eventov-Friedman, S., Katchman, H., Jacob-Hirsch, J., Amariglio, N., Rechavi, G., Margalit, R. and Reisner, Y.: Isolation and Characterization of Nontubular sca-1+lin- Multipotent Stem/Progenitor Cells from Adult Mouse Kidney., J Am Soc Nephrol 17, 3300, 2006
小鼠	小鼠, 雄性, 12-16 周	Renal proximal tubule cells	Collagenase Type 2: 0.1%	DMEM/F-12	Cunningham, R., Xiaofei, E., Steplock, D., Shenolikar, S. and Weinman, E.: Defective PTH Regulation of Sodium-Dependent Phosphate Transport in NHERF-1-/- Renal Proximal Tubule Cells and Wild-Type Cells Adapted to Low-Phosphate Media., Am J Physiol Renal Physiol 289, F933, 2005
小鼠	小鼠, 雄性, 12-16 周	Renal proximal tubule cells	Soybean Trypsin Inhibitor: 0.25%	DMEM/F-12
小鼠	小鼠, 雄性, 2 個月	Cortical collecting duct	Collagenase: 54-178 u/ml	MEM	Sindic, A., Velic, A., Basoglu, C., Hirsch, J., Edemir, B., Kuhn, M. and Schlatter, E.:Uroguanylin and Guanylin Regulate Transport of Mouse Cortical Collecting Duct Independent of Guanylate Cyclase C., Kidney Int 68, 1008, 2005
小鼠	小鼠, 雄性, 2 個月	Cortical collecting duct	Protease: see reference	MEM
小鼠	小鼠, 6 周	Glomerular mesangial cells	Collagenase Type 4: 0.1%	RPMI 1640	Radeke HH, Janssen-Graalfs I, Sowa EN, Chouchakova N, Skokowa J, Loscher F, Schmidt RE, Heeringa P, Gessner JE.: Opposite regulation of type II and III receptors for immunoglobulin G in mouse glomerular mesangial cells and in the induction of anti-glomerular basement membrane (GBM) nephritis, J Biol Chem 277(30), 27535, 2002
小鼠	小鼠, 3-7 個月	Fibroblasts, mesangial, smooth muscle	Trypsin: 0.25%	DMEM	Bradshaw AD, Francki A, Motamed K, Howe C, Sage EH: Primary mesenchymal cells isolated from SPARC-null mice exhibit altered morphology and rates of proliferation, Mol Biol Cell 10, 1569-79, 1999
			Collagenase: see reference
			Soybean Trypsin Inhibitor: .05%
小鼠	小鼠	Epithelial	Collagenase Type 4: 1%	DMEM /F-12	Taub, M: Renal Tubule Cells,Cell & Tissue Culture: Laboratory Procedures Vol. 2,Doyle, A., Griffiths, J., and Newell, D., John Wiley & Sons, Ltd., 14B:6.1, 1995
小鼠	小鼠,單純的 SJL (H-2S)	Proximal tubular epithelial	Deoxyribonuclease I: 15 礸/ml	RPMI 1640	Haverty, T., Kelly, C., Hines, W., Amenta, P., Watanabe, M., Harper, R., Kefalides, N., and Neilson, E.: Characterization of a Renal Tubular Epithelial Cell Line Which Secretes the Autologous Target Antigen of Autoimmune Experimental Interstitial Nephritis, J Cell Biol 107, 1359, 1988
豬	豬, 雜種	Kidney	Collagenase Type 1: 0.1%	HBSS	Heussner, A. and Dietrich, D.: Primary Porcine Proximal Tubular Cells as an Alternative to Human Primary Renal Cells in Vitro: an Initial Characterization., BMC Cell Biol 14, 55, 2013
兔	兔	Renal proximal tubular	Collagenase Type 1: 60 u/ml	DMEM/F12	Nowak, G. and Bakajsova, D.: Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes., J Vis Exp 71, e4301, 2013
兔	兔, 新西蘭	Proximal tubule cells	Collagenase Type 4: 0.005%	DMEM/F-12	Taub, M.: Primary Kidney Proximal Tubule Cells., Methods Mol Biol 290, 231, 2005
兔	兔, 新西蘭	Proximal tubule cells	Soybean Trypsin Inhibitor: 0.00025%	DMEM/F-12
兔	兔, 新西蘭白, 雌性	Nephron segments	Collagenase Type 2: 0.05%	Eagle's MEM	Schafer, J., Watkins, M., Li, L., Herter, P., Haxelmans, S., and Schlatter, E.: A Simplified Method for Isolation of Large Numbers of Defined Nephron Segments, Am J Physiol 273, F650, 1997
兔	兔, 新西蘭白, 雌性	Nephron segments	Deoxyribonuclease I: 50 u/ml	Eagle's MEM
兔	兔, 新西蘭白, 雄性, 1.5 - 2.0 公斤	Duct	Soybean Trypsin Inhibitor: 0.025%	Hank's Solution with calcium and HEPES	Naray-Fejes-Toth, A., and Fejes-Toth, G.: Immunoelection and Culture of Cortical Collecting Duct Cells, J Tiss Cul Meth 13, 179, 1991
兔	兔, 新西蘭掰,雌性, 2-3 公斤	Renal proximal tubules	Deoxyribonuclease I: 70 u/ml	Modified DME-F12	Rodeheaver, D., Aleo, M., and Schnellmann, R.: Differences in Enzymatic and Mechanical Isolated Rabbit Renal Proximal Tubules: Comparison in Long-Term Incubation, In Vitro Cell Dev Biol 26, 898, 1990
兔	兔	Papillary duct	Hyaluronidase: 0.2%	Keri's buffer	Husted, R., Hayashi, M., and Stokes, J.: Characteristics of Papillary Collecting Duct Cells in Primary Culture, Am J Physiol 255, F1160, 1988
兔	(包括, SD 大鼠和Wistar大鼠, 150-225克, 馬, 牛)	Papillary duct	Hyaluronidase: 0.2%	HEPES buffered saline
兔	兔, 新西蘭, 白色, 2-3 公斤	MTALH cells	Collagenase: 0.1%	DMEM	Allen, M., Nakao, A., Sonnenburg, W., Burnatowska-Hledin, M., Speilman, W., and Smith, W.: Immunodissection of Cortical and Medullary Thick Ascending Limb Cells From Rabbit Kidney, Am J Physiol 255, F704, 1988
兔	兔, 新西蘭, 白色, 2-3 公斤	RCCT cells	Collagenase: 0.1%	DMEM
兔	兔, 新西蘭, 白色	Collecting tubule	Trypsin: 0.05%	Kreb's Ringer buffer	Grenier, F., and Smith, W.: Formation of 6-keto-PGF-₁_aby Collecting Tubule Cells Isolated from Rabbit Renal Papillae, Prostaglandins 16, 759, 1978
兔	兔, 新西蘭, 白色, 雌性, 2.5 公斤	Papillary collecting duct	Collagenase: 0.2%	(see reference)	Dworzack, D., and Grantham, J.: Preparation of Renal Papillary Collecting Duct Cells for Study, Nat Neurosci 8, 191, 1975
大鼠	SD大鼠, 胎兒	Fetal kidney	Collagenase Type 4: 0.1%	MEM	Gupta, A., Jadhav, S., Tripathy, N. and Nityanand, S.: Fetal Kidney Cells Can Ameliorate Ischemic Acute Renal Failure in Rats through Their Anti-Inflammatory, Anti-Apoptotic and Anti-Oxidative Effects., PLoS ONE 10, e0131057, 2015
大鼠	Lewis大鼠, 雄性	Renal adipose derived cells	Collagenase Type 1: 0.3%	DMEM	Basu, J., Genheimer, C., Sangha, N., Quinlan, S., Guthrie, K., Kelley, R., Ilagan, R., Jain, D., Bertram, T. and Ludlow, J.: Organ Specific Regenerative Markers in Peri-Organ Adipose: Kidney., Lipids Health Dis 10, 171, 2011
大鼠	大鼠	Proximal tubule	Collagenase Type 2: 0.1%	see reference	Panico, C., Luo, Z., Damiano, S., Artigiano, F., Gill, P. and Welch, W.: Renal Proximal Tubular Reabsorption is Reduced in Adult Spontaneously Hypertensive Rats: Roles of Superoxide and Na+/H+ Exchanger 3., Hypertension 54, 1291, 2009
大鼠	SD大鼠, 雄性, 200-250克	Medullary thick ascending limb	Collagenase: 0.1%	HEPES-saline	Silva, G., Beierwaltes, W., and Garvin, J.: Extracellular ATP stimulates NO production in rat thick ascending limb, Hypertension 47, 563-7, 2006
大鼠	Wistar大鼠, 300克	Renal proximal tubules	Collagenase Type 2: 0.2%	DMEM/F12	Deng Aihua, Miracle Cynthia M, Lortie Mark, Satriano Joseph, Gabbai Francis B, Munger Karen A, Thomson Scott C, Blantz Roland C: Kidney oxygen consumption, carbonic anhydrase, and proton secretion, Am J Physiol/Renal 290, F1009-15, 2006
大鼠	大鼠, Wistar, 雄性, 300-350 克	Renal proximal tubules	Collagenase Type 2: 0.2%	DMEM/F-12	Deng, A., Miracle, C., Suarez, J., Lortie, M., Satriano, J., Thomson, S., Munger, K. and Blantz, R.: Oxygen Consumption in the Kidney: Effects of Nitric Oxide Synthase Isoforms and Angiotensin II., Kidney Int 68, 723, 2005
大鼠	大鼠	Cortical tubule cells	Collagenase: 0.6%	DMEM	Arystarkhova, E., Wetzel R., and Sweadner K.: Distribution and Oligomeric Association of Splice Forms of Na(+)-K(+)-ATPase Regulatory Gamma-subunit in Rat Kidney, Am J Physiol/Renal 282(3), F393, 2002
大鼠	SD大鼠, 雄性 275-300 克	Glomerular mesangial cells	Collagenase Type 1: 250 u/ml	DMEM	Amiri Farhad, Shaw Sean, Wang Xiaodan, Tang Jie, Waller Jennifer L, Eaton Douglas C, Marrero Mario B: Angiotensin II activation of the JAK/STAT pathway in mesangial cells is altered by high glucose, Kidney Int Suppl 61, 1605-16, 2002
大鼠	SD大鼠, 1 周	Cortical collection duct	Collagenase Type 2: 0.15%	DMEM/Ham's F12	Valencia L., Bidet M., Martial S., Sanchez E., Melendez E., Tauc M., Poujeol C., Martin D., Namorado M., Reyes J., and Poujeol P.: Nifedipine-activated Ca(2+) Permeability in Newborn Rat Cortical Collecting Duct Cells in Primary Culture, Am J Physiol Cell Physiol280(5), C1193, 2001
大鼠	SD大鼠, 250-300克	Microvessels	Collagenase Type 2: 0.1%	PSS	Li, N., Teggatz, E., Li, P., Allaire, R., and Zou, A.: Formation and Actions of Cyclic ADP-Ribose in Renal Microvessels, Microvasc Res 60, 149, 2000
大鼠	SD大鼠, 250-300 克	Renal	Collagenase Type 2: 0.2%	see reference	Mattson D., and Wu F.: Nitric Oxide Synthase Activity and Isoforms in Rat Renal Vasculature, Hypertension 35, 337, 2000
大鼠	SD大鼠, 雄性, 5-6 周	Renal tubules	Collagenase: 0.1%	see reference	Miyata, N., Park, F., Li, X., and Cowley, A.: Distribution of Angiotensin AT1 and AT2 Receptor Subtypes in the Rat Kidney, Am J Physiol 277, F437, 1999
大鼠	SD大鼠, 225-250 g	Glomerular mesangial cells	Collagenase Type 1: 250 u/ml	DMEM	Amiri F, Garcia R: Regulation of angiotensin II receptors and PKC isoforms by glucose in rat mesangial cells, Am J Physiol 276, F691-9, 1999
大鼠	SD大鼠, 雄性	Proximal tubule suspensions	Collagenase Type 4: 0.1%	HEPES buffer	Eitle E, Hiranyachattada S, Wang H, Harris PJ: Inhibition of proximal tubular fluid absorption by nitric oxide and atrial natriuretic peptide in rat kidney, Am J Physiol 274, C1075-80, 1998
大鼠	SD大鼠, 雄性	Proximal tubule suspensions	Pronase E: 2 u/ml	HEPES buffer
大鼠	大鼠, SD, 雄性	Nephron segments	Collagenase Type 2: 0.05%	Eagle's MEM	Schafer, J., Watkins, M., Li, L., Herter, P., Haxelmans, S., and Schlatter, E.: A Simplified Method for Isolation of Large Numbers of Defined Nephron Segments, Am J Physiol 273, F650, 1997
大鼠	大鼠, SD, 雄性	Nephron segments	Deoxyribonuclease I: 50 u/ml	Eagle's MEM
大鼠	大鼠, 12-周大	Renal	Collagenase: 0.1%	DMEM	Ishikawa, S., Kusaka, I., Higashiyama, M., Nagasaka, S., Saito, T., Honda, K., and Saito, T.: Cellular Signaling and Proliferative Action of AVP in Mesangium of SHR: Effect of Low Density Lipoprotein, Nat Neurosci 50, 1506, 1996
大鼠	大鼠, 雄性, 150-200 克	Epithelial	Protease: 0.1%	HBSS/CMF	Elliget, K., and Trump, B.: Primary Cultures of Normal Rat Kidney Proximal Tubule Epithelial Cells for Studies of Renal Cell Injury, In Vitro Cell Dev Biol 27, 739, 1991
大鼠	大鼠, 雄性, 150-200 克	Proximal tubule	Protease: 0.1%	HBSS/CMF
大鼠	大鼠, 成年的	Inner medullary collecting duct	Collagenase Type 2: 0.1%	PBS	Brion, L., Schwartz, J., Lachman, H., Zavilowitz, B., and Schwartz, G.: Development of H+ Secretion by Cultured Renal Inner Medullary Collecting Duct Cells, Am J Physiol 257, F486, 1989
大鼠	大鼠, 成年的	Papillae	Collagenase Type 2: 0.1%	PBS
大鼠	Wistar大鼠, 雌性, 100-150 克	Glomerular mesangial	Collagenase: 0.025%	RPMI 1640	Wang, J., Kester, M., and Dunn, M.: The Effects of Endotoxin on Plaet-Activating Factor Synthesis in Cultured Rat Glomerular Mesangila Cells, Biochim Biophys Acta 969, 217, 1988
大鼠	Wistar大鼠, 雄性, 150 - 200 克	Renal target	Collagenase: 1.0% (also 0.7%)	Eagle's MEM	Barlet-Bas, C., Khadouri, C., Marsey, S., and Doucet, A.: Sodium-Independent In VitroInduction of Na+,K+-ATPase by Aldosterone in Renal Target Cells: Permissive Effect of Triiodothyronine, Proc Natl Acad Sci U S A 85, 1707, 1988
大鼠	Wistar大鼠, 雄性, 成年的	Proximal tubules	Collagenase: 0.2%	Krebs-Henseleit buffer	Gesek, F., Wolff, D., and Strandhov, J.: Improved Separation Method for Rat Proximal and Distal Renal Tubules, Am J Physiol 253, F358, 1987
大鼠	Wistar大鼠, 300-400克	Proximal tubules	Collagenase: 0.15 %	Krebs Henseleit solution	Vinay, P., Gougoux, A., and Lemieux, G.: Isolation of a Pure Suspension of Rat Proximal Tubules, Am J Physiol 241, F403, 1981
大鼠	大鼠	Fibroblasts	Trypsin: up to 0.25%	Dulbecco-Vogt MEM	Wallach, D., Anderson, W., and Pastan, I.: Activation of Adenylate Cyclase in Cultured Fibroblasts by Trypsin, J Biol Chem 253, 24, 1978
大鼠	大鼠	Kidney	Trypsin: up to 0.25%	Dulbecco-Vogt MEM

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