原代细胞分离中，针对肠组织涉及到肠 L 细胞，淋巴细胞、固有层单核细胞、肠上皮和黏膜单核细胞、隐窝等等的解离通常会使用酶，如何选择合适的酶来完成对应肠组织的解离，以及原代细胞采集的常规操作， 艾美捷针对肠相关的组织分类解离分种属、不同的细胞、酶组合推荐，推荐浓度、培养基以及参考文献，详细汇总指南：
本技术指南描述了标准实验室程序； 为建立细胞分离方案提供了一种合乎逻辑的实验方法； 并列出了许多组织特异性参考资料，以帮助开发有效的方法。更多其他组织的解离指南，点击查看：各组织分离指南。
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| 种属 | 种属详情 | 细胞名称 | 酶组合推荐，含推荐浓度 | 培养基 | 参考文献 |
| 犬/狗 | Dog, adult | 肠 L 细胞 / Intestinal L-cells | Collagenase Type 1: 75 u/ml | HBSS | Damholt AB, Buchan AM, Kofod H: Glucagon-like-peptide-1 secretion from canine L-cells is increased by glucose-dependent-insulinotropic peptide but unaffected by glucose, Endocrinology 139, 2085-91, 1998 |
| 人 | Human | 淋巴细胞 / Lymphocytes | CLSPA: 0.05%； Deoxyribonuclease I: 0.00075% |
RPMI-1640 | Trapecar, M., Khan, S., Roan, N., Chen, T., Telwatte, S., Deswal, M., Pao, M., Somsouk, M., Deeks, S., Hunt, P., Yukl, S. and Sanjabi, S.: An Optimized and Validated Method for Isolation and Characterization of Lymphocytes from HIV+ Human Gut Biopsies., AIDS Res Hum Retroviruses 33, S31-S39, 2017 |
| 人 | Human | 微血管内皮 / Microvessel endothelial | Collagenase Type 2: 0.2% | HBSS | Kessler, S., Rho, H., West, G., Fiocchi, C., Drazba, J. and de la Motte, C.: Hyaluronan (HA) Deposition Precedes and Promotes Leukocyte Recruitment in Intestinal Inflammation., Clin Transl Sci 1, 57-61, 2008 |
| 人 | Human | 固有层淋巴细胞 / Lamina propria lymphocytes | Collagenase: 20 u/ml | Medium | Ebert, Ellen.: CD2 Activation of Human Lamina Propria Lymphocytes Reduces CD3 Responsiveness., Immunology 117, 71-7, 2006 |
| 人 | Human | 固有层单核细胞 / Lamina propria mononuclear cells | Collagenase: 0.02% | HBSS | Kanai Takanori, Totsuka Teruji, Uraushihara Koji, Makita Shin, Nakamura Tetsuya, Koganei Kazutaka, Fukushima Tsuneo, Akiba Hisaya, Yagita Hideo, Okumura Ko, Machida Utako, Iwai Hideyuki, Azuma Miyuki, Chen Lieping, Watanabe Mamoru: Blockade of B7-H1 suppresses the development of chronic intestinal inflammation, J Immunol 171, 4156-63, 2003 |
| 人 | Human | 肠上皮 / Intestinal epithelial | Collagenase Type 4: 72.5 u/ml | HBSS | Fahlgren, A., Hammarstrom, S., Danielsson, A. and Hammarstrom, M.: Increased Expression of Antimicrobial Peptides and Lysozyme in Colonic Epithelial Cells of Patients with Ulcerative Colitis., Clin Exp Immunol 131, 90, 2003 |
| 人 | Human | 黏膜单核细胞 / Mucosal mononuclear cells | Collagenase Type 3: 0.01%； Deoxyribonuclease I: 0.01%； Soybean Trypsin Inhibitor: 0.01% |
RPMI | Stallmach A, Schaffer F, Hoffmann S, Weber S, Muller-Molaian I, Schneider T, Kohne G, Ecker KW, Feifel G, Zeitz M: Increased state of activation of CD4 positive T cells and elevated interferon gamma production in pouchitis, Gut 43, 499-505, 1998 |
| 小鼠 | Mouse | 固有层淋巴细胞 / Lamina propria lymphocytes | Collagenase Type 4: 0.02%； Deoxyribonuclease I: 0.01% |
HBSS | Emgard, J., Kammoun, H., Garcia-Cassani, B., Chesne, J., Parigi, S., Jacob, J., Cheng, H, Evren, E., Das, S., Czarnewski, P., Sleiers, N. Melo-Gonzalez, F., Kvedaraite, E. Svensson, M. and Scandella, E.: Oxysterol Sensing through the Receptor GPR183 Promotes the Lymphoid-Tissue-Inducing Function of Innate Lymphoid Cells and Colonic Inflammation., Immunity 48, 120-132.e8, 2018 |
| 小鼠 | Mouse, embryonic | 隐窝 / Crypt | Collagenase Type 1: 200 u/ml； Hyaluronidase: 100u/ml |
DMEM | Ren HJ, Zhang CL, Liu RD, Li N, Li XG, Xue HK, Guo Y, Wang ZQ, Cui J and Ming L: Primary Cultures of Mouse Small Intestinal Epithelial Cells using the Dissociating Enzyme Type I Collagenase and Hyaluronidase., Braz J Med Biol Res 50, e5831, 2017 |
| 小鼠 | Mouse | 肠道类器官 / Intestinal organoids | Collagenase Type 4: 500 u/ml； Deoxyribonuclease I: 15 u/ml |
Basal Medium | O'Rourke, K., Ackerman, S., Dow, L. and Lowe, S.: Isolation, Culture, and Maintenance of Mouse Intestinal Stem Cells., Bio Protoc 6, , 2016 |
| 小鼠 | Mouse | 固有层单核细胞 / Lamina propria mononuclear cells | Collagenase Type 3: 400 u/ml； Deoxyribonuclease I: 0.001% |
HBSS | Seo, S., Kuffa, P., Kitamoto, S., Nagao-Kitamoto, H., Rousseau, J., Kim, Y., Nunez, G. and Kamada, N.: Intestinal Macrophages Arising from CCR2(+) Monocytes Control Pathogen Infection by Activating Innate Lymphoid Cells., Nat Commun 6, 8010, 2015 |
| 小鼠 | Mouse | 淋巴细胞 / Lymphocytes | Collagenase Type 1: 100 u/ml | RPMI-1640 | Beura, L., Anderson, K., Schenkel, J., Locquiao, J., Fraser, K., Vezys, V., Pepper, M. and Masopust, D.: Lymphocytic Choriomeningitis Virus Persistence Promotes Effector-Like Memory Differentiation and Enhances Mucosal T cell Distribution., J Leukoc Biol 97, 217, 2015 |
| 小鼠 | Mouse, P4 | 肠神经嵴祖细胞 / Enteric neural crest progenitors | Neutral Protease: 0.5%； Collagenase animal free: 0.05% |
DMEM/F12 | Hotta, R., Stamp, L., Foong, J., McConnell, S., Bergner, A., Anderson, R., Enomoto, H., Newgreen, D., Obermayr, F., Furness, J. and Young, H.: Transplanted Progenitors Generate Functional Enteric Neurons in the Postnatal Colon., J Clin Invest 123, 1182, 2013 |
| 小鼠 | Mouse | 结肠肿瘤类器官 / Colon tumor organoids | Collagenase Type 4: 200 u/ml； Neutral Protease: 0.01% |
DMEM | Xue, X. and Shah, Y.: In Vitro Organoid Culture of Primary Mouse Colon Tumors., J Vis Exp , e50210, 2013 |
| 小鼠 | Mouse | 肠神经元 / Enteric neurons | Collagenase Type 4: 0.1%； Trypsin: 0.05%； Deoxyribonuclease I: 400 u/ml |
DMEM/F12 | Zhang, Y. and Hu, W.: Mouse Enteric Neuronal Cell Culture., Methods Mol Biol 1078, 55-63, 2013 |
| 小鼠 | Mouse | 肠道类器官 / Intestinal organoids | Collagenase Type 1: 800 u/ml； Neutral Protease: 0.013% |
DMEM | Barthel, E., Speer, A., Levin, D., Sala, F., Hou, X., Torashima, Y., Wigfall, C. and Grikscheit, T.: Tissue Engineering of the Intestine in a Murine Model., J Vis Exp 70, e4279, 2012 |
| 小鼠 | Mouse | 树突状，巨噬细胞 / Dendritic, macrophages | Collagenase: 0.15%； Deoxyribonuclease I: 0.004% |
HBSS | Geem, D., Medina-Contreras, O., Kim, W., Huang, C. and Denning T.: Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine., J Vis Exp 63, e4040, 2012 |
| 小鼠 | Mouse | 淋巴细胞 / Lyphocytes | Collagenase Type 4: 0.05%； Neutral Protease: 0.1%； Deoxyribonuclease I: 200 ug/ml |
RPMI 1640 | Ruiz, V., Sachdev, M., Zhang, S., Wen, S. and Moss, S.: Isolating, Immunophenotyping and Ex vivo Stimulation of CD4+ and CD8+ Gastric Lymphocytes During Murine Helicobacter Pylori Infection., J Immunol Methods 384, 157-63, 2012 |
| 小鼠 | Mouse, 2-3 month | 肠神经干细胞 / Enteric neural stem cells | Collagenase: 0.11%； Neutral Protease: 0.1%； Deoxyribonuclease I: 50 u/ml |
M199 | Becker, L., Kulkarni, S., Tiwari, G., Micci, M. and Pasricha, P.: Divergent Fate and Origin of Neurosphere-Like Bodies from Different Layers of the Gut., Am J Physiol Gastrointest Liver Physiol 302, G958-65, 2012 |
| 小鼠 | Mouse, neonatal and adult | 肌间丛 / Myenteric plexus | Collagenase Type 4: 0.025-0.1%； Papain: 10 u/ml； Deoxyribonuclease I: 100 u/ml |
HBSS | Joseph, N., He, S., Quintana, E., Kim, Y., Nunez, G. and Morrison, S.: Enteric Glia are Multipotent in Culture but Primarily Form Glia in the Adult Rodent Gut., J Clin Invest 121, 3398, 2011 |
| 小鼠 | Mouse | 固有层淋巴细胞 / Lamina propria lymphocytes | Collagenase Type 1: 0.3%； Deoxyribonuclease I: 0.01% |
RPMI 1640 | Ito, Y., Kanai, T., Totsuka, T., Okamoto, R., Tsuchiya, K., Nemoto, Y., Yoshioka, A., Tomita, T., Nagaishi, T., Sakamoto, N., Sakanishi, T., Okumura, K., Yagita, H. and Watanabe, M.: Blockade of NKG2D Signaling Prevents the Development of Murine CD4+ T Cell-Mediated Colitis., Am J Physiol Gastrointest Liver Physiol 294, G199, 2008 |
| 小鼠 | Mouse | 肠单核 / Intestinal mononuclear | Collagenase Type 4: 0.1% | RPMI 1640 | Forbes, E., Groschwitz, K., Abonia, J.P., Brandt, E., Cohen, E., Blanchard, C., Ahrens, R., Seidu, L., McKenzie, A., Strait, R., Finkelman, F., Foster, P., Matthaei, K., Rothenberg, M. and Hogan, S.: IL-9- and Mast Cell-Mediated Intestinal Permeability Predisposes to Oral Antigen Hypersensitivity., J Exp Med 205, 897, 2008 |
| 小鼠 | Mouse, Mastomys natalensis | 肠嗜铬细胞 / Enterochromaffin cells | Collagenase: 0.025%； Pronase E: 0.07% |
HBSS | Kidd, M., Modlin, I., Eick, G. and Champaneria, M.: Isolation, Functional Characterization, and Transcriptome of Mastomys Ileal Enterochromaffin Cells., Am J Physiol Gastrointest Liver Physiol 291, G778-91, 2006 |
| 小鼠 | Mouse, 7 week | 间质细胞 / Interstitial cells | Collagenase: 0.04%； Trypsin: 0.02% |
see reference | Goto Kazunori, Matsuoka Satoshi, Noma Akinori: Two types of spontaneous depolarizations in the interstitial cells freshly prepared from the murine small intestine, J Physiol 559, 411-22, 2004 |
| 小鼠 | Mouse, 9-13 day | 卡哈尔间质细胞 / Interstitial cells of Cajal | Collagenase Type 2: 0.13% | HBSS | Ordag Tamas, Redelman Doug, Miller Lisa J, Horvath Viktor J, Zhong Qiao, Almeida-Porada Graca, Zanjani Esmail D, Horowitz Burton, Sanders Kenton M: Purification of interstitial cells of Cajal by fluorescence-activated cell sorting, Am J Physiol Cell Physiol 286, C448-56, 2004 |
| 小鼠 | Mouse, 0-15 day | 卡哈尔间质细胞 / Interstitial cells of Cajal | Collagenase Type 2: 0.13% | Hanks | Lee Young Mee, Kim Byung Joo, Kim Hyun Jin, Yang Dong Ki, Zhu Mei Hong, Lee Kyu Pil, So Insuk, Kim Ki Whan: TRPC5 as a candidate for the nonselective cation channel activated by muscarinic stimulation in murine stomach, Am J Physiol/Gastro 284, G604-16, 2003 |
| 小鼠 | Mouse | 固有层淋巴细胞 / Lamina propria lymphocytes | Collagenase Type 4: 300 u/ml | PBS | Wu Y, Wang X, Csencsits KL, Haddad A, Walters N, Pascual DW: M cell-targeted DNA vaccination, Proc Natl Acad Sci U S A 98, 9318-23, 2001 |
| 小鼠 | Mouse, F14 | 肠间充质 / Intestinal mesenchymal | Collagenase Type 2: 0.03% | HBSS | Sakagami Y, Inaguma Y, Sakakura T, Nishizuka Y: Intestine-like remodeling of adult mouse glandular stomach by implanting of fetal intestinal mesenchyme, Cancer Res 44, 5845-9, 1984 |
| 大鼠 | Rat, postnatal, adult | 肠神经嵴干细胞 / Gut neural crest stem | Collagenase Type 4: 0.1%； Trypsin: 0.025% |
HBSS | Kruger, G., Mosher, J., Bixby, S., Joseph, N., Iwashita, T. and Morrison, S.: Neural Crest Stem Cells Persist in the Adult Gut but Undergo Changes in Self-Renewal, Neuronal Subtype Potential, and Factor Responsiveness., Neuron 35, 657, 2002 |
| 大鼠 | Rat, SD, embryonic | 坐骨神经和肠神经嵴干细胞 / Sciatic nerve and gut neural crest stem | Collagenase Type 4: 0.025%； Trypsin: 0.005%； Deoxyribonuclease I: 0.05% |
HBSS | Bixby, S., Kruger, G., Mosher, J., Joseph, N. and Morrison, S.: Cell-Intrinsic Differences Between Stem Cells from Different Regions of the Peripheral Nervous System Regulate the Generation of Neural Diversity., Neuron 35, 643, 2002 |
| 大鼠 | Rat, male, 18-24 day | 肠上皮 / Intestinal epithelial | Collagenase Type 1: 0.1% | DMEM | Quaroni A, Wands J, Trelstad RL, Isselbacher KJ: Epithelioid cell cultures from rat small intestine. Characterization by morphologic and immunologic criteria, J Cell Biol 80, 248-65, 1979 |
