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Isolation of Epithelial Cells from Mouse Gastrointestinal Tract for Western Blot or RNA Analysis
从小鼠胃肠道中分离上皮细胞进行WB或RNA分析(根据Kulkarni等人1985年发表的试验改进)   

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Abstract

The gastrointestinal (GI) tract is lined by a single layer of epithelial cells which function in secretion, absorption, and digestion. In addition, most GI tract tumors develop from epithelial cells (carcinomas). This protocol describes isolation of the surface epithelium from the underlying stroma, muscular layer and submucosa in the GI tract. In this protocol, epithelial cell adhesions are weekend by chelating Ca +2 ions followed by mechanical separation of the cells by vortexing. Analysis of protein levels and gene expression patterns in isolated epithelial cells versus whole GI tissue minimizes the potential for confounding contributions from contaminating stromal cells.

Keywords: Intestine tissue(内部组织), Colon tissue(结肠组织), Cell isolation(细胞的分离), Protein(蛋白), Mouse(鼠标)

Materials and Reagents

  1. 4% sodium hypochlorite (Sigma-Aldrich, catalog number: 239305 )
  2. Phosphate buffered saline (PBS) (Mediatech, Cellgro®, catalog number: 21-031 )
  3. KCl
  4. NaCl
  5. KH2PO4
  6. Na2HPO4
  7. EDTA
  8. DTT
  9. Aprotinin (USB, catalog number: 9087-70-1 )
  10. Leupeptin (MP, catalog number: 195623 )
  11. Pepstatin (MP, catalog number: 195368 )
  12. PMSF (Sigma-Aldrich, catalog number: 329-98-5 )
  13. Ethanol
  14. 0.04% sodium hypochlorite (see Recipes)
  15. 100x protease inhibitor (store at -20 °C) (see Recipes)
  16. Solution B (see Recipes)
  17. 1x lysis buffer (see Recipes)

Equipment

  1. CO2 chamber for mice
  2. Dissecting scissors (one medium size and one small size for dissecting mice)
  3. Forceps (2)
  4. 15-ml conical tubes
  5. 1.5-ml eppendorf tubes
  6. 3ml Insulin syringe
  7. Vortex
  8. Centrifuge compatible with 15-ml conical

Procedure

  1. Make sure to prepare all the solutions including PBS at least 1 h before the start of killing the mice and store them on ice so they are cold when you start to work with them.
  2. Sacrifice the mouse (18-25 grams) by CO2 asphyxiation according to American Veterinary Medical Association (AVMA) guidelines on Euthanasia, 2007 (https://www.avma.org/KB/Policies/Documents/euthanasia.pdf). Briefly, first carbon dioxide’s anesthetic properties are used by exposing rodents to a slowly rising concentration of carbon dioxide until they become unconscious. A chamber fill rate of 20%, or 1/5 th of the cage volume per minute is recommended. Slower, uncoordinated movements occur after about 1 min, animals stop moving after about 2 min, and unconsciousness occurs after about 2.5 min. After unconsciousness is achieved, flow can be increased to hasten death. Two-step euthanasia is used to ensure death. Once the animal has lost consciousness and is unresponsive to a toe pinch, a follow-up method is utilized. This can include decapitation with sharp scissors, cervical dislocation, or thoracotomy.
  3. Open the abdomen and dissect the gastrointestinal tract from the stomach to anus by pulling gently on the stomach and removal of the mesentry (the peritoneal folding around the intestine which connects the intestinal tract to the dorsal abdominal wall. It contains fat, blood vessels and lymph nodes).
  4. Cut the intestine into pieces of interest (Jejunum, Ileum and large intestine).
  5. Keep the pieces in cold PBS on ice and work with them one at a time.
  6. Flush the content of the intestine with cold PBS to a 3 m clean it using l insulin syringe. Alternatively, invert the intestine on a glass rod and swirl in cold PBS
  7. Wash intestinal pieces in cold PBS and keep them until you are done with all pieces (to synchronize your work).
  8. Incubate pieces in 50- 100 ml of 0.04% sodium hypochlorite on ice for 15 min (this works well using a small beaker). This step removes bacterial contaminants.
  9. During this time prepare three 15-ml conical tubes for every tissue piece and put 5-10 ml of solution B in each tube (if you are using glass rods. 10 ml will be necessary). Mark the tubes and leave them on ice.
  10. Remove intestinal pieces from the sodium hypochlorite and rinse in PBS.
  11. Put the intestinal pieces in the 15 ml conical containing solution B for 15 min on ice.
  12. Remove solution B and add 5 ml PBS (or solution B). I do this by holding the intestinal piece with a long forceps, decanting the solution B then adding back the intestinal piece and add PBS.
  13. Vortex for 15 sec.
  14. Take the intestinal pieces and put them in another 15 ml conical with solution B.
  15. Repeat steps 11-14 twice (you will have 3 tubes per piece).
  16. Suspend the cells from all tubes for every piece and mix them in one tube.
  17. Centrifuge at 1,000 rpm (1,000 x g, 10 min at 4 °C).
  18. Take the fluid off and suspend the cells in lysis buffer (5 ml for Jejunum and Ileum or 1 ml for colon) for total cell lysate or Trizol (Invitrogen) 1 ml for RNA extraction.

Recipes

  1. 0.04% sodium hypochlorite
    1 ml 4% sodium hypochlorite
    99 ml PBS
  2. Solution B
    2.7 mM KCl 0.2 g or 2.7 ml 1 M KCl
    150 mM NaCl 8.77 g 50 ml 3 M NaCl solution
    1.2 mM KH2PO4 0.16 g
    680 mM Na2HPO4 9.65 g
    1.5 mM EDTA 0.44 g 3 ml 0.5 M EDTA solution
    0.5 mM DTT 0.08 g 1 ml 0.5M DTT (add fresh)
    Bring to 1 L in ddH2O
  3. 100x protease inhibitor (store at -20 °C)
    10 mg aprotinin
    10 mg leupeptin
    10 mg pepstatin
    174.2 mg PMSF
    Bring to 10 ml in absolute ethanol
    Vortex well before using
  4. 1x lysis buffer
    5x reporter lysis buffer     2 ml
    ddH2O                          7.9 ml
    100x protease inhibitor    100 μl

Acknowledgments

This protocol was modified from a crypt isolation procedure reported by Kulkarni and Yielding (1985). This work was supported by RO1 CA10922 from the National Cancer Institute and P20s RR15563 and RR016475.

References

  1. Kulkarni, M. S. and Yielding, K. L. (1985). DNA damage and repair in epithelial (mucous) cells and crypt cells from isolated colon. Chem Biol Interact 52(3): 311-318.
  2. Zeineldin, M., Cunningham, J., McGuinness, W., Alltizer, P., Cowley, B., Blanchat, B., Xu, W., Pinson, D. and Neufeld, K. L. (2012). A knock-in mouse model reveals roles for nuclear Apc in cell proliferation, Wnt signal inhibition and tumor suppression. Oncogene 31(19): 2423-2437.

简介

胃肠(GI)道由在分泌,吸收和消化中起作用的单层上皮细胞排列。 此外,大多数胃肠道肿瘤从上皮细胞(癌)发展。 该协议描述了表面上皮从下面的基质,肌肉层和胃肠道中的粘膜下层的分离。 在该协议中,上皮细胞粘附是周末通过螯合Ca +2离子,然后通过涡旋机械分离的细胞。 分离的上皮细胞与整个GI组织中蛋白质水平和基因表达模式的分析使来自污染性基质细胞的混杂贡献的潜力最小化。

关键字:内部组织, 结肠组织, 细胞的分离, 蛋白, 鼠标

材料和试剂

  1. 4%次氯酸钠(Sigma-Aldrich,目录号:239305)
  2. 磷酸盐缓冲盐水(PBS)(Mediatech,Cellgro ,目录号:21-031)
  3. KCl
  4. NaCl
  5. KH 2 PO 4
  6. Na HPO 4
  7. EDTA
  8. DTT
  9. 抑肽酶(USB,目录号:9087-70-1)
  10. 亮抑酶肽(MP,目录号:195623)
  11. 胃酶抑素(MP,目录号:195368)
  12. PMSF(Sigma-Aldrich,目录号:329-98-5)
  13. 乙醇
  14. 0.04%次氯酸钠(参见配方)
  15. 100x蛋白酶抑制剂(储存在-20°C)(参见配方)
  16. 解决方案B(参见配方)
  17. 1x裂解缓冲液(见配方)

设备

  1. 小鼠的CO 2腔室
  2. 解剖剪刀(一种中型和一种小型解剖小鼠)
  3. 镊子(2)
  4. 15毫升锥形管
  5. 1.5 ml eppendorf管
  6. 3ml胰岛素注射器
  7. 涡流
  8. 离心机兼容15-ml锥形

程序

  1. 确保在开始杀死老鼠之前至少1小时准备所有包括PBS的溶液,并将其存储在冰上,以便在开始使用它们时感冒。
  2. 根据美国兽医协会(AVMA)关于Euthanasia的指南,通过CO 2窒息牺牲小鼠(18-25克)(https://www.avma.org/KB/Policies/Documents /euthanasia.pdf)。 简言之,通过将啮齿动物暴露于缓慢升高的二氧化碳浓度直到它们变得无意识,来使用第一二氧化碳的麻醉特性。 腔室填充率为20%,或每分钟笼体积的1/5 推荐的。在约1分钟后出现较慢的,未协调的运动,在约2分钟后动物停止移动,并且在约2.5分钟后出现无意识。在实现无意识之后,可以增加流量以加速死亡。两步安乐死用于确保死亡。一旦动物已经失去意识并且对脚趾捏力不响应,则使用随访方法。这可以包括使用锋利的剪刀,颈椎脱位或胸廓切开术进行头晕
  3. 打开腹部,通过在胃上轻轻地拉动并移除肠系膜(腹膜折叠在肠道周围,将肠道连接到背腹壁,将胃肠道从胃分离到肛门),其包含脂肪,血管和淋巴节点)。
  4. 将肠切成感兴趣的部分(空肠,回肠和大肠)
  5. 将这些碎片放在冰上的冷PBS中,一次处理一次。
  6. 用冷PBS将肠内容物冲洗至3 m,用胰岛素注射器清洗。或者,将玻璃棒上的肠倒置,并在冷PBS中旋转
  7. 在冷PBS中清洗肠道碎片并保存,直到您完成所有的碎片(同步您的工作)。
  8. 孵育片在50-100毫升的0.04%次氯酸钠冰上15分钟(这使用小烧杯很好)。 此步骤可去除细菌污染物。
  9. 在此期间,为每个组织片准备三个15-ml锥形管,并在每个管中放入5-10ml溶液B(如果使用玻璃棒,则需要10ml)。 标记管子,将它们留在冰上。
  10. 从次氯酸钠中取出肠片,在PBS中冲洗
  11. 将肠片在15ml含有锥形的溶液B中在冰上15分钟
  12. 取出溶液B,加入5ml PBS(或溶液B)。 我这样做是通过用长镊子握住肠段,倾析溶液B,然后加回肠段并加入PBS。
  13. 涡旋15秒。
  14. 取肠片,并用溶液B将它们放入另一个15 ml锥形瓶中
  15. 重复步骤11-14两次(每件3个试管)。
  16. 从每个管的所有管悬浮细胞,并将它们混合在一个管
  17. 以1,000rpm(1,000×g/min,4℃,10分钟)离心
  18. 取出液体并将细胞悬浮在裂解缓冲液(对于空肠和Ileum为5ml或对于结肠为1ml)中用于全细胞裂解物或将Trizol(Invitrogen)用于RNA提取1ml。

食谱

  1. 0.04%次氯酸钠
    1ml 4%次氯酸钠
    99 ml PBS
  2. 解决方案B
    2.7mM KCl 0.2g或2.7ml 1M KCl
    150mM NaCl 8.77g 50ml 3MNaCl溶液
    1.2mM KH 2 PO 4 0.16g
    680mM Na 2 HPO 4 9.65g
    1.5mM EDTA 0.44g 3ml 0.5M EDTA溶液
    0.5mM DTT 0.08g 1ml 0.5M DTT(加入新鲜)
    在ddH <2> O中加入1 L
  3. 100x蛋白酶抑制剂(-20℃保存)
    10mg抑肽酶
    10mg亮肽素 10mg胃蛋白酶
    174.2mg PMSF
    在无水乙醇中达到10ml 在使用
    之前充分涡旋
  4. 1x裂解缓冲液
    5x报告裂解缓冲液   2 ml
    ddH 2 O                          7.9 ml
    100x蛋白酶抑制剂   100μl

致谢

该方案由Kulkarni和Yielding(1985)报道的隐窝分离程序修改。 这项工作得到国家癌症研究所的RO1 CA10922和P20s RR15563和RR016475的支持。

参考文献

  1. Kulkarni,M.S.and Yielding,K.L。(1985)。 DNA损伤并在上皮(粘液)细胞和孤立结肠的隐窝细胞中修复。 Chem Biol Interact 52(3):311-318。
  2. Zeineldin,M.,Cunningham,J.,McGuinness,W.,Alltizer,P.,Cowley,B.,Blanchat,B.,Xu,W.,Pinson,D.and Neufeld,K.L。 敲入小鼠模型揭示了核Apc在细胞增殖,Wnt信号抑制和肿瘤抑制中的作用 。 Oncogene 31(19):2423-2437。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Zeineldin, M. and Neufeld, K. (2012). Isolation of Epithelial Cells from Mouse Gastrointestinal Tract for Western Blot or RNA Analysis. Bio-protocol 2(22): e292. DOI: 10.21769/BioProtoc.292.
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