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Scratch Wound Healing Assay
划痕实验   

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Abstract

The scratch wound healing assay has been widely adapted and modified to study the effects of a variety of experimental conditions, for instance, gene knockdown or chemical exposure, on mammalian cell migration and proliferation. In a typical scratch wound healing assay, a “wound gap” in a cell monolayer is created by scratching, and the “healing” of this gap by cell migration and growth towards the center of the gap is monitored and often quantitated. Factors that alter the motility and/or growth of the cells can lead to increased or decreased rate of “healing” of the gap (Lampugnani, 1999). This assay is simple, inexpensive, and experimental conditions can be easily adjusted for different purposes. The assay can also be used for a high-throughput screen platform if an automated system is used (Yarrow and Perlman, 2004).

Materials and Reagents

  1. Human MDA-MB-231 cell line (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. Phosphate buffered saline (PBS) (Life Technologies, Invitrogen™, catalog number: 14190-144 )
  5. Glutaraldehyde (Sigma-Aldrich, catalog number: G6257 )
  6. Ethanol (Sigma-Aldrich, catalog number: 459836 )
  7. Crystal violet (Sigma-Aldrich, catalog number: C3886 )

Equipment

  1. BD Falcon 24-well tissue culture plate (Fisher Scientific, catalog number: 08-772-1H; BD Biosciences, catalog number: 353226 )
  2. Rainin pipet tips (1 ml) (Mettler-Toledo, catalog number: GPS-L1000 )
  3. Cell culture incubator: 37 °C and 5% CO2

Software

  1. Photoshop or ImageJ (http://rsb.info.nih.gov/ij/download.html)

Procedure

  1. Grow cells in DMEM supplemented with 10% FBS.
  2. Seed cells into 24-well tissue culture plate at a density that after 24 h of growth, they should reach ~70-80% confluence as a monolayer.
  3. Do not change the medium. Gently and slowly scratch the monolayer with a new 1 ml pipette tip across the center of the well. While scratching across the surface of the well, the long-axial of the tip should always be perpendicular to the bottom of the well. The resulting gap distance therefore equals to the outer diameter of the end of the tip. The gap distance can be adjusted by using different types of tips. Scratch a straight line in one direction.
  4. Scratch another straight line perpendicular to the first line to create a cross in each well.
  5. After scratching, gently wash the well twice with medium to remove the detached cells.
  6. Replenish the well with fresh medium.
    Note: Medium may contain ingredients of interest that you want to test, e.g., chemicals that inhibit/promote cell motility and/or proliferation.
  7. Grow cells for additional 48 h (or the time required if different cells are used).
  8. Wash the cells twice with 1x PBS, then fix the cells with 3.7% paraformaldehye for 30 min.
  9. Stain the fixed cells with 1% crystal violet in 2% ethanol for 30 min.
  10. Take photos for the stained monolayer on a microscope. Set the same configurations of the microscope when taking pictures for different views of the stained monolayer. The gap distance can be quantitatively evaluated using software such as Photoshop or ImageJ (http://rsb.info.nih.gov/ij/download.html). To reduce variability in results, it’s suggested that multiple views of each well should be documented, and each experimental group should be repeated multiple times.

Acknowledgments

This protocol was developed in the Department of Immunology, Scripps Research Institute, La Jolla, CA, USA and adapted from Lampugnani (1999) and Yarrow et al. (2004). 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. Lampugnani, M. G. (1999). Cell migration into a wounded area in vitro. Methods in Mol Biol 96: 177-182.
  3. Yarrow, J. C., Perlman, Z. E., Westwood, N. J., Mitchison, T. J. (2004). A high-throughput cell migration assay using scratch wound healing, a comparison of image-based readout methods. BMC Biotechnol 4: 21.

简介

划伤伤口愈合测定法已经广泛适应和修改以研究多种实验条件例如基因敲低或化学暴露对哺乳动物细胞迁移和增殖的影响。 在典型的伤口创伤愈合测定中,通过刮擦产生细胞单层中的"伤口间隙",并且监测并常常定量通过细胞迁移和朝向间隙中心的生长的该间隙的"愈合"。 改变细胞运动性和/或生长的因素可导致间隙"愈合"的增加或减少的速率(Lampugnani,1999)。 该测定简单,便宜,并且可以容易地针对不同目的调整实验条件。 如果使用自动化系统,该测定也可以用于高通量筛选平台(Yarrow和Perlman,2004)。

材料和试剂

  1. 人MDA-MB-231细胞系(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. 磷酸盐缓冲盐水(PBS)(Life Technologies,Invitrogen TM,目录号:14190-144)
  5. 戊二醛(Sigma-Aldrich,目录号:G6257)
  6. 乙醇(Sigma-Aldrich,目录号:459836)
  7. 结晶紫(Sigma-Aldrich,目录号:C3886)

设备

  1. BD Falcon 24孔组织培养板(Fisher Scientific,目录号:08-772-1H; BD Biosciences,目录号:353226)
  2. Rainin移液管吸头(1ml)(Mettler-Toledo,目录号:GPS-L1000)
  3. 细胞培养孵育器:37℃和5%CO 2/h

软件

  1. Photoshop或ImageJ( http://rsb.info.nih.gov/ij/download.html

程序

  1. 在补充有10%FBS的DMEM中生长细胞
  2. 将种子细胞以24小时生长后的密度接种到24孔组织培养板中,它们应当作为单层达到约70-80%汇合。
  3. 不要更换介质。轻轻地和慢慢地划伤单层,使用新的1毫升移液管尖端穿过孔的中心。当刮擦孔的表面时,尖端的长轴应始终垂直于孔的底部。因此,所得到的间隙距离等于尖端的端部的外径。间隙距离可以通过使用不同类型的尖端来调节。沿一个方向划一条直线。
  4. 划出另一条垂直于第一条直线的直线,在每个井中创建一个十字
  5. 擦伤后,用培养基轻轻冲洗孔两次,以除去分离的细胞
  6. 用新鲜培养基补充孔。
    注意:中等可能含有您想测试的感兴趣的成分,例如抑制/促进细胞运动和/或增殖的化学物质。
  7. 生长细胞额外48小时(或如果使用不同的细胞所需的时间)。
  8. 用1x PBS洗涤细胞两次,然后用3.7%多聚甲醛固定细胞30分钟
  9. 用1%结晶紫在2%乙醇中染色固定的细胞30分钟
  10. 在显微镜上为染色的单层拍照。当为染色单层的不同视图拍摄照片时,设置相同的显微镜配置。间隙距离可以使用诸如Photoshop或ImageJ( http://rsb。 info.nih.gov/ij/download.html )。为了减少结果的变异性,建议对每个孔的多个视图进行记录,并且每个实验组应重复多次。

致谢

该方案在美国加利福尼亚州La Jolla的Scripps研究所免疫学系中开发,并改编自Lampugnani(1999)和Yarrow等人。 (2004)。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由Jiing-Dwan Lee博士资助[参见Chen (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. Lampugnani,M.G。(1999)。 体外细胞迁移到受伤区域 。 < em> Methods in Mol Biol   96:177-182。
  3. Yarrow,J.C.,Perlman,Z.E.,Westwood,N.J.,Mitchison,T.J。(2004)。 使用刮痕伤口愈合的高通量细胞迁移测定,基于图像的读出方法的比较。 BMC Biotechnol 4:21.
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Chen, Y. (2012). Scratch Wound Healing Assay. Bio-protocol 2(5): e100. DOI: 10.21769/BioProtoc.100.
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Carl Lee
internal medicine of University of Utah
Is the cell in growth status or starvation status, means the concentration of FBS should be 10% or 0.1%-1%, when the cell treated with drugs? Thank you.
12/1/2014 2:13:09 PM Reply
Yanling Chen
The Scripps Research Institute

The protocol described here is for what you called "growth status", and you can modify it according your experimental requirements-it really depends on your assay goals and cell type. As you probably know, many cells (even cancer cells) in low serum media won't grow as well as in high (i.e., 10%) serum conditions. If your purpose is to maximize the effect of a drug under low serum conditions, you can try your cells of interest at lower FBS for sure, but the assay conditions should be empirically determined (for a better "combination" of assay settings).

12/2/2014 7:31:42 AM


larita salv
imcb
Hi,
I'm having some troubles reproducing some knockdown results.
Sometimes, independently of scratch width, my scratched cells migrate very poorly and after more than 48h wound is still open and cells alive.
Does it ever happen to you? Any advice?
Another question: does sodium pyruvate in the medium affect wound healing?
Thanks a lot!
8/4/2013 10:06:01 PM Reply
Yanling Chen
The Scripps Research Institute

Outcome of the "healing" process very much depends on the cell type, i.e., those that migrate aggresively/spread out quickly probably will heal the gap much faster, such as the example cell line mentioned in the protocol. Also, the "healing" process is resulted from not only cell migrating, but also cell growth. finally, please select enough views and repeat the same assay several times-just to reduce variability in readouts so that they are objective and reliable (statistically).
And I don't think sodium pyruvate would have negative effect on would healing.
Hope that these could help.

8/6/2013 7:20:19 PM


Lisha Chen
SINANO
Hi, I would like to know whether I should coat some ECM like Matrigel or other proteins? Some articles I have read recommed protein coating before experiment. Thank you!
5/28/2013 8:38:13 PM Reply
Yanling Chen
The Scripps Research Institute

You may coat the surface with some proteins as long as the protein layer is even and uniform. For Matrigel, I think it's more difficult to scratch the surface to generate a gap, and the cells may grow into the EMC and stay there (not grow to "health" the "gap"). Please try and find a better and creative way for your experiments.

5/29/2013 7:18:24 AM


Lisha Chen
SINANO

Thank you! I have another question. Should I change the medium to the serum free medium before I scratch the line? Since I need to detect a cytokine activity. When I detect the proliferation activation, I always change to a serum free medium to starve the cell before add the cytokine. Thank you!

5/31/2013 12:09:35 AM


Yanling Chen
The Scripps Research Institute

I think switching to serum-free medium should not be a problem as long as your cells can survive and grow well (for some time) in the serum-free medium. But please keep in mind that some cell lines need serum to maintain normal metabolism and other functions such as spreading out or moving etc.

6/4/2013 6:20:12 AM


rose luo
university
is this assay can evaluate the nitric oxide-induced endothelial cells dysfunction?Thank you a lots!
5/6/2013 11:56:15 PM Reply
Yanling Chen
The Scripps Research Institute

This assay is used to study the outcomes of a specific treatment of cells for cell migrating and growth. I don't have laboratory experience with assays of nitric oxide induction of endothetial cells. If you are to test cell migration and growth as an experimental endpoint, you may want to modity the assay design for that purpose.

5/9/2013 2:14:25 PM


Carmen Toro
UASLP
Hi,
I would like to make this assay (modifying something) and then trypsinize the cells in order to obtain single cells to do patch clamp on those cells. Is that possible? How can I to obtain two groups: migrating and no migrating cells? Could you please give me some suggestion?
Thank you in advance.
5/8/2012 11:36:22 AM Reply
Yanling Chen
The Scripps Research Institute

For the patch clamp assay, you probably have to try it because I do not have this information.
For seperating/obtaining migrating and non-migrating cells (again sorry I don't have this info). You might want to try a different method, e.g. cell invasion assay using matrigel and then retrieve the migrated cells from the gel? Just my suggestion.

6/1/2012 1:34:55 PM


why there is need to stain with crystal violet? can we visualize the cells inside the wells or we have to take out the round coverslip?
3/25/2012 9:01:52 PM Reply
Yanling Chen
The Scripps Research Institute

Staining with crystal violet (other other dyes of your choice) would make visualization easier; also better for taking pictures.
And if you need to grow cells on coverslips (that may be coated with reagents of your interest), you will need to modify/optimize the protocols.

6/1/2012 1:34:37 PM


nor hidayah
UKM

i wanna ask..after staining with crystal violet solution...is there need to remove the staining solution before taking photo?ASAP

4/3/2014 5:05:37 AM