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Transwell Cell Migration Assay Using Human Breast Epithelial Cancer Cell
Transwell小室检测人乳腺上皮癌细胞迁移   

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Abstract

Transwell migration assays have been widely used for studying the motility of different types of cells including metastatic cancer cells. The assay is also useful in screens for compounds that act as chemoattractants or inhibitors of chemotaxis for cells. The assay employs a permeable layer of support, usually a tissue-culture-treated microporous membrane, which is positioned between two compartments that mimic two different sets of microenvironments for cell survival/growth. Cells on one side of the membrane, when sensing chemoattractants placed on the other side of the compartment that diffuses through the membrane, can migrate through the pores in the membrane towards the source of the chemoattractants. Cells that migrate across the membrane can be quantified by fixing and counting. Human breast epithelial adenocarcinoma MD-231 cells grow relatively fast and are metastatic. The MB-231 cell line is used here to describe the procedures of an in vitro cell migration assay using the transwell apparatus.

Materials and Reagents

  1. Human MDA-MB-231 cell (ATCC, catalog number: HTB-26 ™)
  2. Dulbecco's modified eagle medium (DMEM) (Life Technologies, Invitrogen™, catalog number: 10313-021 )
  3. Fetal bovine serum (FBS) (ATCC, catalog number: 30-2020 ™)
  4. Trypsin-EDTA (Life Technologies, Invitrogen™, catalog number: 25200-056 )
  5. Trypsin inhibitor (soybean) (Life Technologies, Invitrogen™, catalog number: 17075-029 )
  6. Phosphate buffered saline (PBS) (Life Technologies, Invitrogen™, catalog number: 14190-144 )
  7. Collagen I (Sigma-Aldrich, catalog number: C7661 ) or Fibronectin (BD Biosciences, catalog number: 354008 )
  8. Glutaraldehyde (Sigma-Aldrich, catalog number: G6257 )
  9. Ethanol (Sigma-Aldrich, catalog number: 459836 )
  10. Crystal violet (Sigma-Aldrich, catalog number: C3886 )
  11. TCC-formulated Leibovitz's L-15 Medium (ATCC, catalog number: 30-2008 ™)

Equipment

  1. Corning® Transwell® polycarbonate membrane inserts (Sigma-Aldrich, catalog number: CLS3421 ) or Millicell Cell Culture Inserts (EMD Millipore, catalog number: PI8P01250 )
  2. Cotton swabs
  3. Cell culture incubator: 37 °C and 5% CO2

Procedure

  1. Carry MB-231 cells in DMEM with 10% FBS (use L-15 medium if needed).
  2. Wash cells twice with 1x PBS and trypsinize.
  3. Add 0.5 mg/ml Trypsin inhibitor in PBS to inactivate an equal volume of Trypsin. Aspirate cells by pipetting up and down gently (Note: It is important to break down into individual cells as much as possible).
  4. Gently spin down the cells. Wash cells two times with DMEM containing 0.5% FBS to remove trace amounts of trypsin and inhitibor. Resuspend the cells in DMEM with 0.5% FBS and count.
  5. Prepare the transwell compartments, 24-well format, with 8 μm pore size insert:
    1. To the lower compartment, add 2.6 ml of DMEM with 0.5% FBS containing 40 μg ml-1 Collagen I.
    2. Add the transwell insert to the well by merging the bottom of the insert into the medium in the lower compartment.
    Note: Ensure that no air bubbles are trapped between the insert membrane and the medium.
  6. To the upper compartment, gently add 1 x 105 cells from step 4.
  7. Incubate the cells in the transwell plate at 37 °C and 5% CO2 for 2.5 h. This allows cells to migrate toward the underside of the insert filter.
  8. After 2.5 h, carefully take the insert out. Cells that do not migrate through the pores and therefore remain on the upper side of the filter membrane need to be gently removed with a cotton swab. Gently wipe the upper side of the filter membrane with a cotton swab to remove the cell debris. We recommend that use each clean cotton swab for one wipe only, in one direction and do not swipe in back-and-forth movement. The cotton swab can be slightly moisturized with ddH2O as needed but be sure to remove any excess water. Several wipes may be needed to completely remove any cell debris on the membrane.
  9. Fix the cells on the lower side of the insert filter quickly with 5% glutaraldehyde for 10 min.
  10. Next, stain cells on the lower side of the insert filter with 1% crystal violet in 2% ethanol for 20 min.
  11. Remove excess crystal violet by quickly merging the insert in ddH2O for 3 to 4 sec. Drain excess water from the side of the insert using a cotton swab. Dry the insert membrane.
  12. Count the number of cells on the lower side of the filter under a microscope. Randomly choose different views and take average counting.
  13. The same experimental procedure should be performed for control groups without chemoattractants. Each migration condition should be tested with replicates.

Acknowledgments

This protocol was developed in the Department of Immunology, Scripps Research Institute, La Jolla, CA, USA and adapted from Katoh et al. (2006) and Nakamizo et al. (2005). The work was funded by NIH grants CA079871 and CA114059, and Tobacco-Related Disease, Research Program of the University of California, 15RT-0104 to Dr. Jiing-Dwan Lee  [see Chen et al. (2009)].

References

  1. Chen, Y., Lu, B., Yang, Q., Fearns, C., Yates, J. R., 3rd and Lee, J. D. (2009). Combined integrin phosphoproteomic analyses and small interfering RNA--based functional screening identify key regulators for cancer cell adhesion and migration. Cancer Res 69(8): 3713-3720.
  2. Katoh, H., Hiramoto, K. and Negishi, M. (2006). Activation of Rac1 by RhoG regulates cell migration. J Cell Sci 119(Pt 1): 56-65.
  3. Nakamizo, A., Marini, F., Amano, T., Khan, A., Studeny, M., Gumin, J., Chen, J., Hentschel, S., Vecil, G., Dembinski, J., Andreeff, M. and Lang, F. F. (2005). Human bone marrow-derived mesenchymal stem cells in the treatment of gliomas. Cancer Res 65(8): 3307-3318.

简介

Transwell迁移测定已经广泛用于研究不同类型的细胞(包括转移性癌细胞)的运动性。该测定法还可用于筛选充当化学趋化剂或细胞趋化性抑制剂的化合物。该测定使用可渗透的支持层,通常是组织培养处理的微孔膜,其位于模拟用于细胞存活/生长的两种不同组的微环境的两个区室之间。膜的一侧上的细胞,当感测放置在通过膜扩散的隔室的另一侧上的化学引诱物时,可以通过膜中的孔向化学引诱物的来源迁移。穿过膜迁移的细胞可以通过固定和计数来定量。人乳腺上皮腺癌MD-231细胞生长相对较快并且是转移性的。在此使用MB-231细胞系来描述使用transwell装置的体外细胞迁移测定的程序。

材料和试剂

  1. 人MDA-MB-231细胞(ATCC,目录号:HTB-26 TM)
  2. Dulbecco's改良的Eagle培养基(DMEM)(Life Technologies,Invitrogen TM,目录号:10313-021)
  3. 胎牛血清(FBS)(ATCC,目录号:30-2020 TM)
  4. 胰蛋白酶-EDTA(Life Technologies,Invitrogen TM,目录号:25200-056)
  5. 胰蛋白酶抑制剂(大豆)(Life Technologies,Invitrogen TM,目录号:17075-029)
  6. 磷酸盐缓冲盐水(PBS)(Life Technologies,Invitrogen TM,目录号:14190-144)
  7. 胶原I(Sigma-Aldrich,目录号:C7661)或纤连蛋白(BD Biosciences,目录号:354008)
  8. 戊二醛(Sigma-Aldrich,目录号:G6257)
  9. 乙醇(Sigma-Aldrich,目录号:459836)
  10. 结晶紫(Sigma-Aldrich,目录号:C3886)
  11. TCC配制的Leibovitz's L-15培养基(ATCC,目录号:30-2008 TM)

设备

  1. (Sigma-Aldrich,目录号:CLS3421)或Millicell细胞培养插入器(EMD Millipore,目录号:PI8P01250)的缓冲液中。
  2. 棉签
  3. 细胞培养孵育器:37℃和5%CO 2/h

程序

  1. 携带MB-231细胞在含10%FBS的DMEM中(如果需要,使用L-15培养基)
  2. 用1x PBS洗涤细胞两次并胰蛋白酶消化
  3. 在PBS中加入0.5mg/ml胰蛋白酶抑制剂以灭活等体积的胰蛋白酶。 通过轻轻地上下吸液吸取细胞(注意:尽可能细分为单个细胞是非常重要的)。
  4. 轻轻地旋转细胞。 用含有0.5%FBS的DMEM洗涤细胞两次,以除去痕量的胰蛋白酶和抑制剂。 重悬细胞在含有0.5%FBS的DMEM中并计数
  5. 准备transwell隔室,24孔格式,具有8μm孔径插入:
    1. 向下部室加入2.6ml含有40μg/ml胶原I的0.5%FBS的DMEM。
    2. 通过将插入物的底部合并到下室中的培养基中,将孔中的插入物添加到孔中。
    注意:确保插入膜和介质之间没有气泡。
  6. 到上隔室,轻轻加入来自步骤4的1×10 5个细胞
  7. 孵育transwell板中的细胞在37℃和5%CO 2下2.5小时。这允许细胞迁移到插入过滤器的下侧。
  8. 2.5小时后,小心取出插件。不通过孔迁移并因此保留在滤膜上侧的细胞需要用棉签轻轻去除。用棉签轻轻擦拭滤膜的上侧,以除去细胞碎片。我们建议仅使用每个干净的棉签进行一次擦拭,在一个方向上,不要在前后运动中滑动。根据需要,棉签可以用ddH 2 O轻微润湿,但必须除去任何过量的水。可能需要几次擦拭以完全去除膜上的任何细胞碎片
  9. 用5%戊二醛快速固定插入过滤器下侧的细胞10分钟
  10. 接下来,用1%结晶紫在2%乙醇中的插入过滤器下侧的染色细胞20分钟
  11. 通过快速合并ddH <2> O中的插入物3至4秒除去过量的结晶紫。使用棉签从插件的侧面排出多余的水。干燥插入膜。
  12. 在显微镜下计数过滤器下侧的细胞数。随机选择不同的视图并进行平均计数。
  13. 应该对没有化学引诱物的对照组进行相同的实验程序。每个迁移条件都应该用重复测试。

致谢

该方案在美国加利福尼亚州La Jolla的Scripps研究所的免疫学部开发,并改编自Katoh等人。 (2006)和Nakamizo等人(2005)。该工作由NIH资助,CA079871和CA114059,以及加利福尼亚大学的烟草相关疾病研究计划,15RT-0104给Jiing-Dwan Lee博士[见Chen等人]。 (2009)]。

参考文献

  1. Chen,Y.,Lu,B.,Yang,Q.,Fearns,C.,Yates,J.R.,3rd和Lee,J.D。(2009)。 组合整合素磷蛋白分析和基于小干扰RNA的功能筛选确定了癌细胞粘附的关键调节因子, 。 Cancer Res 69(8):3713-3720。
  2. Katoh,H.,Hiramoto,K。和Negishi,M。(2006)。 RhoG对Rac1的激活调节细胞迁移。 111(Pt 1):56-65。
  3. Nankamizo,A.,Marini,F.,Amano,T.,Khan,A.,Studeny,M.,Gumin,J.,Chen,J.,Hentschel,S.,Vecil,G.,Dembinski, Andreeff,M。和Lang,FF(2005)。 人骨髓间充质干细胞在治疗胶质瘤中的应用 Cancer Res 65(8):3307-3318。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Chen, Y. (2012). Transwell Cell Migration Assay Using Human Breast Epithelial Cancer Cell. Bio-protocol 2(4): e99. DOI: 10.21769/BioProtoc.99.
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Young-Sang Kim
PuKyong National University
Hello, I performed transwell migration assay with HT29 cells.
when I take pictures, for counting cells, I cannot distinguish the cells and pores(see the picture).
So, I need help for staining and washing and how to take a picture using microscope.
Other papaers with same experiment, their figures are very clear, when i read papers.
please Help me.
1/28/2016 11:55:46 PM Reply
7/31/2014 9:54:42 PM Reply
xl w
shanghai chempartner
Hi, is the membrane is permeable to collagen? If so, the concentration of collagen will equilibrate between the up and low compartment. How collagen can still work as chemattractants?
Thank you!
2/19/2014 4:04:46 AM Reply
Yanling Chen
The Scripps Research Institute

This experimental setup allows gradient of chemoattractant be established, which is the "driving force" for migration. We don't know how long it takes to "equilibrate" if that would happen eventually, time is also a determining factor here, I think.

2/21/2014 8:29:57 AM


Lisha Chen
SINANO
Hi, I would like to know why the collagen I were added in the medium in the lower compartment. I saw some experimental procedures say the uupper chambers should be pre-coated with rat collagen type I.What's the function of the rat tail collagen I in this assay? Thank you!
7/2/2013 10:56:38 PM Reply
Yanling Chen
The Scripps Research Institute

Collagen is added as a chemoattractant; you may use other chemoattractants of your choice.

7/19/2013 7:03:50 AM


Lisha Chen
SINANO

Thank you!I have another question. Is the 2% ethanol in crystal violet solution necessary? If I use the solution that crystal violet dissovled in ddH2O, can it work?

8/13/2013 1:18:07 AM


Yanling Chen
The Scripps Research Institute

I guess that should work too as long as you can get it dissolved well.

8/13/2013 7:30:49 AM


It would be really helpful if you could be more specific about the volume in which we need to dispense the cell in each insert . Just cell number does not help. Also what will be the volume of agonist in the lower chamber and cell in upper chamber in a 96 well plate?
10/16/2012 4:01:16 AM Reply
Hi there,

I have few questions regarding this essay. First, MDA231 cells in my hand didn't migrate to the centre of the insert. After migration (24 h), I saw lot of cells at the rim of the insert. I tried to change the way i added cells, volume of media and cell numbers in the insert. But still none of these worked. I always used 10 % FBS as the chemoattractant (I used comoplete media for this). My first try was ok. Cells migrated evenly through the membrane. But after that I could not get the same result.
Could you please suggest me what would be the problem here?

Many thanks
10/9/2012 8:20:21 PM Reply
Yanling Chen
The Scripps Research Institute

1. Please make sure that the bottom of the insert, when immersed in medium, has no small bubbles trapped underneath (especially the middle area). The microporous membrane should be soaked evenly in medium.
2. Please add the volumes of media/cell according to manufacturer's manual, which keeps the inside/outside media at certain levels.
3. Avoid swirling medium when adding cells to the insert.
4. The transwell plate should be kept steady when incubated, reduce moving it around or shaking it.
5. For your experiments, you may also way to try different cell seeding densities, and/or a shorter migration time.
Hope these can help.

10/17/2012 9:34:08 PM


Will MDA-MB-231 cells migrate/invade with FBS and not EGF? I have read several papers that imply this cell line will not invade/migrate without it. Thanks!
1/24/2012 6:14:35 AM Reply
Yanling Chen
The Scripps Research Institute

In the author's hands, EGF has never been required for successful migration assay using MB231 cells. Some researchers even carry out this assay using medium without either FBS or EGF. Please see reference: PLoS One. 2010 Dec 30;5(12):e15940.

1/28/2012 6:07:03 PM