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[Bio101] Tumor Cell Invasion Assay
[Bio101] 肿瘤细胞侵袭试验   

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Abstract

Cell invasion assays have been used to study the interactions between tumor cells and the extracellular matrix (ECM), which not only provides a structural scaffold for the cell, but also contains various biological factors for the survival and growth of the cell. ECM gel contains the basic components of the ECM that provides a structural support for the cell to grow and move. Cells can secrete enzymes that degrade certain components of the ECM to move towards chemoattractants, or to simply establish niches for growth. Metastatic tumor cells often show more invasiveness to the ECM gel due to their higher motility and/or enzymatic activity for degrading ECM components. This protocol describes a tumor cell invasion assay to study the interactions between tumor cells and the ECM.

Materials and Reagents

  1. ECM gel (Sigma-Aldrich, catalog number: E1270 )
  2. Human MDA-MB-231 cell line (ATCC, catalog number: HTB-26 ™)
  3. Dulbecco's modified eagle medium (DMEM) (Life Technologies, Invitrogen™, catalog number: 10313-021 )
  4. Fetal bovine serum (FBS) (ATCC, catalog number: 30-2020 ™)
  5. Trypsin-EDTA (Life Technologies, Invitrogen™, catalog number: 25200-056 )
  6. Phosphate buffered saline (PBS) (Life Technologies, Invitrogen™, catalog number: 14190-144 )
  7. Glutaraldehyde (Sigma-Aldrich, catalog number: G6257 )
  8. Ethanol (Sigma-Aldrich, catalog number: 459836 )
  9. Crystal violet (Sigma-Aldrich, catalog number: C3886 )

Equipment

  1. Cell culture incubator: 37 °C and 5% CO2
  2. Millicell cell culture inserts of 12 mm diameter 8 μm pores (Merck KGaA, catalog number: PI8P01250 )
  3. Cotton swabs

Procedure

  1. Grow cells in DMEM supplemented with 10% FBS.
  2. Thaw ECM gel overnight at 4 °C and keep on ice.
  3. Chill Millicell insert and plate to 4 °C, keep on ice.
  4. Dilute ECM gel in ice-cold, serum-free DMEM to a final concentration of 2 mg/ml.
    Note: ECM gel final concentration may vary, depending on the cell type studied.
  5. Add 100 μl ECM gel from step 4 into the upper compartment of the Millicell insert.
  6. Immediately incubate the plate, with insert and ECM gel inside, at 37 °C for 2 h. This allows the liquid ECM gel to solidify.
  7. Wash cells with 1x PBS and trypsinized the cells. Wash cells 2 times with serum-free DMEM and resuspend in DMEM at 5 x 105 cell/ml.
  8. To the well of the plate (lower compartment), add 1.5 ml of DMEM supplemented with 10% FBS as attractant. Position the insert into the well, with the bottom of the insert merged in medium.
  9. Gently add 1 x 105 cells from step 7 to the upper compartment of the insert.
  10. Incubate the plate at 37 °C for 24 h.
  11. After the incubation period, take the insert out carefully. Cells and the gel in the upper compartment of the insert need to be gently removed by gently wiping the upper side of the membrane with a cotton swab. We recommend to 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.
  12. Fix the cells on the lower side of the insert membrane with 5% glutaraldehyde for 10 min, followed by staining with 1% crystal violet in 2% ethanol for additional 20 min.
  13. Quickly merge the insert in ddH2O for 3-4 sec to remove excess dye, and immediately drain excess water using a cotton swab. Dry the insert completely.
  14. Count the number of cells on the lower side of the filter under a microscope. Randomly choose different views and take average counting.

Acknowledgments

This protocol was developed in the Department of Immunology, Scripps Research Institute, La Jolla, CA, USA and adapted from Repesh (1989) and Valster 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  (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. Hauck, C. R., Hsia, D. A., Puente, X. S., Cheresh, D. A. and Schlaepfer, D. D. (2002). FRNK blocks v-Src-stimulated invasion and experimental metastases without effects on cell motility or growth. EMBO J 21(23): 6289-302.
  3. Repesh, L. A. (1989). A new in vitro assay for quantitating tumor cell invasion. Invasion and Metastasis 9(3): 192-208.
  4. Valster, A., Tran, N. L., Nakada, M., Berens, M. E., Chan, A. Y. and Symons, M. (2005). Cell migration and invasion assays. Methods 37(2): 208-215.

简介

细胞侵袭测定已经用于研究肿瘤细胞和细胞外基质(ECM)之间的相互作用,其不仅为细胞提供结构支架,而且还包含用于细胞存活和生长的各种生物因子。 ECM凝胶含有ECM的基本组分,为细胞生长和移动提供结构支持。 细胞可分泌降解ECM的某些组分以趋向化学引诱物的酶,或简单地建立用于生长的壁龛。 转移性肿瘤细胞通常对ECM凝胶显示更多的侵袭性,这是由于它们对于降解ECM组分的较高的运动性和/或酶活性。 该协议描述了肿瘤细胞侵袭实验,以研究肿瘤细胞和ECM之间的相互作用。

材料和试剂

  1. ECM凝胶(Sigma-Aldrich,目录号:E1270)
  2. 人MDA-MB-231细胞系(ATCC,目录号:HTB-26 TM)
  3. Dulbecco's改良的Eagle培养基(DMEM)(Life Technologies,Invitrogen TM,目录号:10313-021)
  4. 胎牛血清(FBS)(ATCC,目录号:30-2020 TM)
  5. 胰蛋白酶-EDTA(Life Technologies,Invitrogen TM,目录号:25200-056)
  6. 磷酸盐缓冲盐水(PBS)(Life Technologies,Invitrogen TM,目录号:14190-144)
  7. 戊二醛(Sigma-Aldrich,目录号:G6257)
  8. 乙醇(Sigma-Aldrich,目录号:459836)
  9. 结晶紫(Sigma-Aldrich,目录号:C3886)

设备

  1. 细胞培养孵育器:37℃和5%CO 2/h
  2. 12mm直径8μm孔的Millicell细胞培养插入物(Merck KGaA,目录号:PI8P01250)
  3. 棉签

程序

  1. 在补充有10%FBS的DMEM中生长细胞
  2. 解冻ECM凝胶在4℃过夜,并保持在冰上
  3. Chill Millicell插入并平板至4°C,保持冰。
  4. 在冰冷的无血清DMEM中稀释ECM凝胶至终浓度为2mg/ml 注意:ECM凝胶最终浓度可能会有所不同,具体取决于研究的细胞类型。
  5. 从步骤4添加100微升ECM凝胶到Millicell插入物的上隔室
  6. 立即孵化板,插入和ECM凝胶内,在37℃下2小时。 这允许液体ECM凝胶固化
  7. 用1x PBS洗涤细胞并胰蛋白酶化细胞。 用无血清DMEM洗涤细胞2次,并以5×10 5个细胞/ml重悬于DMEM中。
  8. 向板(下室)的孔中加入1.5ml补充有10%FBS的DMEM作为引诱剂。 将插入件放入井中,插入件的底部在介质中合并。
  9. 从步骤7轻轻添加1×10 5 细胞到插入物的上隔室
  10. 将板在37℃孵育24小时
  11. 孵化期后,小心插入。细胞和插入物上隔室中的凝胶需要通过用棉签轻轻擦拭膜的上侧轻轻地除去。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
  12. 用5%戊二醛将细胞固定在插入膜的下侧10分钟,然后用在2%乙醇中的1%结晶紫再染色20分钟。
  13. 快速合并插入ddH 2 O O 3-4秒,以去除多余的染料,并立即使用棉签排出多余的水。完全干燥插件。
  14. 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。

致谢

该方案在美国加利福尼亚州La Jolla的Scripps研究所的免疫学系中开发,并改编自Repesh(1989)和Valster等人(2005)。该工作由NIH授予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, (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. Hauck,C.R.,Hsia,D.A.,Puente,X.S.,Cheresh,D.A。和Schlaepfer,D.D。(2002)。 FRNK阻断v-Src刺激的侵袭和实验性转移,而不影响细胞运动或生长。 EMBO J 21(23):6289-302。
  3. Repesh,L.A。(1989)。 用于定量肿瘤细胞侵袭的新的体外试验。 Invasion and Metastasis 9(3):192-208。
  4. Valster,A.,Tran,N.L.,Nakada,M.,Berens,M.E.,Chan,A.Y。和Symons,M。(2005)。 细胞迁移和侵袭测定。方法 37(2 ):208-215。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Chen, Y. (2012). Tumor Cell Invasion Assay. Bio-protocol Bio101: e101. DOI: 10.21769/BioProtoc.101;
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