搜索

[Bio101] Isolation of Endothelial Cells from Mice
[Bio101] 从小鼠中分离内皮细胞   

下载 PDF 引用 收藏 4 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

Endothelial cells line the entire circulatory system, from the heart to the smallest capillary. In this protocol, attaining and maintaining vitality of the organ is critical. Minimizing the processing time between removing the organ and beginning the digestions will improve yields as cells are more viable and are not under hypoxic conditions for long. For this protocol to be most effective, mice should be 5-6 weeks of age. Older mice result in lower yields of endothelial cells. In our hands, as few as 5 mice can result in good yields.

Materials and Reagents

  1. Endothelial cells (EC)
  2. Endothelial cell growth supplement (ECGS) (Sigma-Aldrich, catalog number: E2759 )
  3. Collagenase I (Life Technologies, InvitrogenTM, catalog number: 17100-017 )
  4. Anti-CD31 antibody (BD Biosciences, PharmingenTM, catalog number: 550274 )
  5. BSA
  6. DMEM
  7. Fetal calf serum (FCS)
  8. Fetal bovine serum (FBS)
  9. EDTA
  10. Phosphate buffered saline (PBS)
  11. Antibiotic
  12. Heparin
  13. F12 medium
  14. Isoflourane
  15. 70% ethanol
  16. Dynabeads
  17. M199
  18. Gelatin
  19. Pre-warmed digestion solution
  20. Digestion solution (see Recipes)
  21. Culture media (see Recipes)

Equipment

  1. Centrifuges
  2. Sterile culture hood
  3. Magnet

Procedure

  1. Harvesting of mouse lungs:
    1. Anesthetize the mice until drowzy. We usually use an inhaled anesthetic such as isoflurane.
    2. Sterilize the skin of the mice with 70% ethanol (sterilize instruments as well).
    3. Cut across the mouse, below but parallel to the diaphragm, and then cut up through the ribs to expose the thoracic cavity.
    4. Excise the lungs rapidly and place them in chilled DMEM on ice. Once all of the lungs have been collected, move to a sterile culture hood for the remainder of the procedure.
    5. Mince lung tissues, rinse several times to remove blood and incubate with 3 ml digestion solution at 37 °C for 45 min (3 x 15 min with pipetting between). Spin down.
    6. Mince the lung tissue finely (into pieces approximately 1 mm 3) and place into 5 ml of digestion buffer at 37 °C.
    7. Agitate the mixture constantly by hand, changing the digestion mixture every 5 min. To do this, spin down the fragments and remove the supernatant, replace the media using 5 ml of pre-warmed digestion solution and continue mixing.
    8. Add 1/10 volume of serum to the digestion solution. This will inactivate the enzymes and allow the cells to attach.
    9. Plate the cells on tissue culture plastic for 1 h. This pre-plate allows for the adhesion of fibroblasts but not endothelial cells (EC).
    10.  Remove the unattached cells and spin down at 200 x g for 5 min, then directly go to the enrichment protocol.

  2. Enriching for endothelial cells:
    Coat dynabeads with antibody to isolate EC: Add 25 μl of dynabeads (1 x 107) into a 1.5 ml centrifuge tube and wash the beads twice with 1 ml of PBS, add 100 μg anti-CD31 antibody in 100 μl PBS to dynabeads and incubate for 24 h at 4 °C with slow mixing. Collect the anti-CD31-coated dynabeads with the dynal magnet. Discard the supernatant while the tube is in the dynal magnet and wash the dynabeads with PBS once for 10 min at 4 °C, for 30 min at 4 °C and finally overnight at 4 °C. Collect the dynabeads using the magnet and discard the supernatant. Resuspend in 500 μl PBS/0.1% BSA and store at 4 °C at a final concentration of 2 x 107 beads/ml.

  3. Isolating endothelial cells using magnetic beads:
    1. After centrifuging the cells (200 x g for 5 min), add 15% FBS+M199. Wash the cells 3x with 15% FBS+M199 (37 °C), and centrifuge at 200 x g for 5 min to pellet cells.
    2. Resuspend in 1-2% FBS+M199 to a final concentration of 10-40 x 106 cells/ml.
    3. Add 1: 10 ratio of beads to endothelial cells (endothelial cells comprise approximately 0.02% of total cells).
    4. Rotate bead-pellet at 4 °C for 30 min. Place tube on magnet for 2-3 min, remove supernatant, and resuspend beads in 2 ml of M199. Repeat for a total of four washes.
    5. To liberate cells from anti-CD31 antibody coated beads, add 5 mM EDTA in PBS and rotate at 37 °C for 10 min.
    6. After 4 washes, place the beads in culture media. For culture of mouse EC, coat the plate with 0.2% gelatin for 20 min prior to plating cells.

Recipes

  1. Digestion solution
    1 mg/mI (250 U) collagenase I
    10 mg/mI BSA
    1 U/mI d ispase
    All dissolved in DMEM.
  2. Culture medium
    20% (v/v) FCS
    10 μg/ml ECGS
    100 U/ml antibiotic
    20 U/ml heparin
    All dissolved in F12 medium

References

  1. Dong, Q. G., Bernasconi, S., Lostaglio, S., De Calmanovici, R. W., Martin-Padura, I., Breviario, F., Garlanda, C., Ramponi, S., Mantovani, A. and Vecchi, A. (1997). A general strategy for isolation of endothelial cells from murine tissues. Characterization of two endothelial cell lines from the murine lung and subcutaneous sponge implants. Arterioscler Thromb Vasc Biol 17(8): 1599-1604.
  2. Sobczak, M., Dargatz, J. and Chrzanowska-Wodnicka, M. (2010). Isolation and culture of pulmonary endothelial cells from neonatal mice. J Vis Exp (46).

简介

内皮细胞分布整个循环系统,从心脏到最小的毛细血管。 在这个协议中,获得和保持器官的活力是至关重要的。 最小化去除器官和开始消化之间的处理时间将提高产量,因为细胞更可行并且长时间不处于低氧条件下。 为了使该方案最有效,小鼠应当是5-6周龄。 老年小鼠导致内皮细胞的产量降低。 在我们手中,少至5只小鼠可以产生良好的产量。

材料和试剂

  1. 内皮细胞(EC)
  2. 内皮细胞生长补充剂(ECGS)(Sigma-Aldrich,目录号:E2759)
  3. 胶原酶I(Life Technologies,Invitrogen TM ,目录号:17100-017)
  4. 抗CD31抗体(BD Biosciences,Pharmingen TM ,目录号:550274)
  5. BSA
  6. DMEM
  7. 胎牛血清(FCS)
  8. 胎牛血清(FBS)
  9. EDTA
  10. 磷酸盐缓冲盐水(PBS)
  11. 抗生素
  12. 肝素
  13. F12中等
  14. 异氟烷
  15. 70%乙醇
  16. Dynabeads
  17. M199
  18. 明胶
  19. 预热消解液
  20. 消解解决方案(参见配方)
  21. 培养基(见配方)

设备

  1. 离心机
  2. 无菌培养罩
  3. 磁铁

程序

  1. 小鼠肺收获:
    1. 麻醉小鼠,直到昏昏欲睡。 我们通常使用吸入麻醉剂,如异氟烷
    2. 消毒小鼠的皮肤用70%乙醇(消毒仪器以及)。
    3. 切过小鼠,在下面但平行于隔膜,然后通过肋切开暴露胸腔。
    4. 快速切除肺并将其置于冰冷的DMEM中。 一旦   所有的肺都已收集,移动到无菌培养罩 对于剩余的程序。
    5. 剁碎肺组织,冲洗 数次以去除血液并与3ml消化溶液孵育 在37℃下45分钟(3×15分钟,之间用移液管)。 向下旋转。
    6. 将肺组织细碎(切成大约1mm 3),并置于37℃下的5ml消化缓冲液中。
    7. 用手搅拌混合物,改变消化 混合物每5分钟。 要做到这一点,旋转碎片,删除 上清液,用5ml预热消化代替培养基 溶液并继续混合
    8. 向消化溶液中加入1/10体积的血清。 这将使酶失活并允许细胞附着。
    9. 板细胞在组织培养塑料上1小时。 这个前板 允许成纤维细胞而不是内皮细胞(EC)的粘附
    10.  移除未附加的储存格,并以200 x g 旋转5分钟,然后直接前往富集协议。

  2. 富集内皮细胞:
    用抗体分离EC的外壳dynabeads:向1.5ml离心管中加入25μldynabeads(1×10 7),并用1ml PBS洗涤珠子两次,加入100μg抗CD31抗体 在100μlPBS中的dynabeads并孵育 24小时,4℃,缓慢混合。用dynal磁体收集抗CD31包被的dynabeads。弃去上清液,同时管在dynal磁铁中,用PBS洗涤dynabeads一次在4℃下10分钟,在4℃下30分钟,最后在4℃下过夜。收集dynabeads使用磁铁和丢弃上清液。重悬于500μlPBS/0.1%BSA中并在4℃下以2×10 7珠/ml的终浓度储存。
  3. 使用磁珠分离内皮细胞:
    1. 离心细胞(200×g/g,5分钟)后,加入15%FBS + M199。洗  细胞用15%FBS + M199(37℃)洗3次,并在200×g离心5分钟  min以沉淀细胞
    2. 重悬于1-2%FBS + M199中至终浓度为10-40×10 6个细胞/ml。
    3. 加入珠与内皮细胞(内皮细胞占总细胞的约0.02%)的1:10比例
    4. 旋转珠粒在4℃下30分钟。 将管放在磁铁上2-3 min,去除上清液,并将珠重悬于2ml M199中。 重复   共四次洗涤。
    5. 为了从抗CD31抗体包被的珠释放细胞,加入PBS中的5mM EDTA,并在37℃下旋转10分钟。
    6. 洗涤4次后,将珠置于培养基中。 文化的 小鼠EC,在铺板前用0.2%明胶涂覆板20分钟 细胞。

食谱

  1. 消化解决方案
    1mg/ml(250U)胶原酶I 10mg/ml BSA
    1 U/ml d ispase
    所有溶于DMEM。
  2. 培养基
    20%(v/v)FCS
    10μg/ml ECGS
    100U/ml抗生素
    20 U/ml肝素
    所有溶于F12培养基中

参考文献

  1. Dong,QG,Bernasconi,S.,Lostaglio,S.,De Calmanovici,RW,Martin-Padura,I.,Breviario,F.,Garlanda,C.,Ramponi,S.,Mantovani,A。和Vecchi, (1997)。 从鼠组织中分离内皮细胞的一般策略。 来自鼠肺和皮下海绵植入物的两种内皮细胞系的表征。 Arterioscler Thromb Vasc Biol 17(8):1599-1604。
  2. Sobczak,M.,Dargatz,J。和Chrzanowska-Wodnicka,M。(2010)。 来自新生小鼠的肺内皮细胞的分离和培养。 (46)。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Pang, H. (2012). Isolation of Endothelial Cells from Mice. Bio-protocol Bio101: e205. DOI: 10.21769/BioProtoc.205;
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。

jie zhang
zhejiang university
Hello
I was wondering why you change M199 into F12 in culture medium? While M199 is suitable for endothelial cells.
Thank you.
Jie
12/21/2015 10:38:20 PM Reply
Hua Li
UniSA
Hi ,

what is the meduim recipe? THank you very much.
and how many passages can do based on this protocol?

which passage will be good for tube-formation assay.

Thank you so much.

Hua
10/19/2013 11:15:08 PM Reply
10/8/2012 3:37:04 PM Reply
Bio-protocol Editorial Team
bio-protocol.org

Thanks for being interested in this protocol. Unfortunately, it is out of reach for us to provide general consulting service. Here, we only are able to answer specific questions regarding this protocol. So, if you have questions regarding this protocol, please be specific.

Thanks,
Bio-protocol team

10/8/2012 10:42:30 PM


Hello,

I was wondering if you could tell me approximately how many endothelial cells you would get from a typical mouse lung. I'm trying to estimate how many endothelial cells are typically found in an adult and embryonic mouse.
Thanks so much!

Janet
2/2/2012 12:17:46 AM Reply
Huan Pang
Molecular Pharmacology, Albert Einstein College of Medicine, USA

Hi,

Normally you could get aorund 10000~50000 endothelial cells from 5 mice in 5-6 weeks old. This is we roughly got in the experiment, but a lot of cells are lost during the isolation and purification precedure.

2/3/2012 1:20:17 PM


BM H
UBC

Hello,

I find the very last steps somewhat confusing:
"4) Rotate bead-pellet at 4°C for 30 minutes. Place tube on magnet for 2-3minutes, remove supernatant, and resuspend beads in 2ml of M199. Repeat for a total of four washes.
5) To liberate cells from anti-CD31 antibody coated beads, add 5mM EDTA in PBS and rotate at37°C for 10 minutes.
6) After 4 washes, place the beads in culture media. For culture of mouse EC, coat the plate with 0.2% gelatin for 20 minutes prior to plating cells."

After washing the beads four times in step (4), you are liberating the cells with 5mM EDTA, so I assume the cells are now in the supernatant, and no longer attached to the beads. So why do you wash the beads again four more times in step (6) and then plate the beads in the culture medium?

Thank you.

8/1/2014 9:53:12 AM