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[Bio101] Cell Adhesion Assay
[Bio101] 细胞吸附试验

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Abstract

Cell adhesion, the binding of a cell to the extracellular matrix (ECM), other cells, or a specific surface, is essential for the growth and survival of the cell and also its communication with other cells. The process of cell adhesion involves a range of biological events such as three-dimensional re-organization of the cytoskeleton, biochemical reactions in the cell, and changes in molecules on the surface of the cell. Cancer cells, especially the highly metastatic types, are believed to have enhanced adhesion ability that often facilitates the migration of the cells to a new site to establish new tumors in the body. Cell adhesion assay is therefore often used to evaluate the metastatic ability of cancer cells. In addition, the assay can also be used to assess the effect of certain treatment (e.g., exposure to chemicals) on the ability of cells to adhere. A modified cell adhesion assay protocol is described here for studying the interactions between cells and extracellular materials.

Materials and Reagents

  1. Hela cells (ATCC, catalog number: CCL-2 ™)
  2. MTT cell proliferation assay kit (ATCC, catalog number: 30-1010K ™)
  3. Collagen I (Sigma-Aldrich, catalog number: C7661 )
  4. Dulbecco's modified eagle medium (DMEM) (Life Technologies, Invitrogen™, catalog number: 10313-021 )
  5. Fetal bovine serum (FBS) (ATCC, catalog number: 30-2020 ™)
  6. 0.5 M EDTA solution (pH 8.0) (Life Technologies, Invitrogen™/Ambion®, catalog number: AM9260G )
  7. Bovine serum albumin (BSA) (Life Technologies, Invitrogen™, catalog number: 15561-020 )
  8. Phosphate buffered saline (PBS) (Life Technologies, Invitrogen™, catalog number: 14190-144 )

Equipment

  1. Corning 96-well polystyrene plate (Fisher Scientific, catalog number: 07-200-91 ; Corning Incorporated, catalog number: 3598 )
  2. Cell culture incubator: 37 °C and 5% CO2
  3. Spectrophotometer that can measure absorption at 570 nm with 96-well format

Procedure

  1. Grow the Hela cells in DMEM supplemented with 10% FBS.
  2. Prepare 40 μg/ml Collagen I solution in PBS, store at 4 °C; prepare 0.1% BSA solution in DMEM.
  3. Coat the 96-well plate (30 μl/well) with the Collagen I solution at 4 °C.
  4. After 12 h of coating, remove the Collagen I solution and air-dry the plate at room temperature in the tissue-culture hood.
  5. Deprive cells of serum for 8 h before the adhesion assay. To do so, wash cells three times with serum-free DMEM and grow them in DMEM.
  6. Use 10 mM EDTA in DMEM to detach the cells and then observe them under a microscope to confirm complete dissociation of the cells, which would take ~10 min.
  7. Wash cells twice with DMEM to remove EDTA, resuspend cells at 2 x 105 cells/ml in DMEM with 0.1% BSA.
  8. For cell-substratum adhesion assay, add 100 μl cell suspension (from step 7) to each of the Collagen I-coated wells. Incubate the plate at 37 °C for 20 min to allow the cells to adhere to the surface.
  9. Add 100 μl DMEM to each well to wash off any non-adherent cells, wash four times.
    Note: To achieve consistency, always add/remove DMEM gently with multi-channel pipetter for multiple wells.
  10. After washing, add DMEM with 10% FBS and incubate the cells at 37 °C for 4 h for recovery.
  11. Add 10 μl of MTT substrate to each well and continue incubation for an additional 2 h at 30 °C.
  12. Next, lyse the MTT-treated cells in 100 µl DMSO (or other lysis buffer of choice) and measure absorbance at 570 nm on a spectrophotometer (see Note 1).

Notes

  1. Consider including the following reference group for monitoring each step of the procedure: Wells not coated with Collagen I; wells not washed with DMEM; wells not added with cells; wells not added with MTT (background for MTT assay).

Acknowledgments

This protocol was developed in the Department of Immunology, Scripps Research Institute, La Jolla, CA, USA and adapted from Chen et al. (2009), Humphries et al. (1998) and Mobley and Shimizu (2001). 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. Humphries, M. (1998). Curr Protoc Cell Biol  9.1.1-9.1-11.
  3. Mobley, J. and Shimizu, Y. (2001). Curr Protoc Immunol  Chapter 7: Unit 7.28.

简介

细胞粘附,细胞与细胞外基质(ECM),其他细胞或特定表面的结合对于细胞的生长和存活以及其与其它细胞的连通是必需的。细胞粘附的过程涉及一系列生物事件,例如细胞骨架的三维重组,细胞中的生化反应和细胞表面上分子的变化。癌细胞,特别是高度转移的类型,被认为具有增强的粘附能力,通常促进细胞迁移到新的位置以在体内建立新的肿瘤。因此,细胞粘附测定常用于评价癌细胞的转移能力。此外,该测定也可以用于评价某些治疗(例如,暴露于化学品)对细胞粘附能力的影响。本文描述了修饰的细胞粘附测定方案,用于研究细胞和细胞外物质之间的相互作用。

材料和试剂

  1. Hela细胞(ATCC,目录号:CCL-2 TM)
  2. MTT细胞增殖测定试剂盒(ATCC,目录号:30-1010K TM)
  3. 胶原I(Sigma-Aldrich,目录号:C7661)
  4. Dulbecco's改良的Eagle培养基(DMEM)(Life Technologies,Invitrogen TM,目录号:10313-021)
  5. 胎牛血清(FBS)(ATCC,目录号:30-2020 TM)
  6. 0.5M EDTA溶液(pH 8.0)(Life Technologies,Invitrogen TM/Ambion ,目录号:AM9260G)
  7. 牛血清白蛋白(BSA)(Life Technologies,Invitrogen TM,目录号:15561-020)
  8. 磷酸盐缓冲盐水(PBS)(Life Technologies,Invitrogen TM,目录号:14190-144)

设备

  1. Corning 96孔聚苯乙烯板(Fisher Scientific,目录号:07-200-91; Corning Incorporated,目录号:3598)
  2. 细胞培养孵育器:37℃和5%CO 2/h
  3. 分光光度计,可以测量在570 nm的吸光度96孔格式

程序

  1. 在补充有10%FBS的DMEM中生长Hela细胞
  2. 在PBS中制备40μg/ml胶原I溶液,在4℃储存; 制备0.1%BSA的DMEM溶液
  3. 在4℃下用胶原I溶液涂覆96孔板(30μl/孔)。
  4. 涂覆12小时后,取出胶原蛋白溶液,并在室温下在组织培养罩中空气干燥平板。
  5. 剥离细胞的血清8小时后粘附测定。为此,用无血清DMEM洗涤细胞三次,并在DMEM中生长
  6. 使用10mM EDTA在DMEM中分离细胞,然后在显微镜下观察它们以确认细胞的完全解离,这将需要〜10分钟。
  7. 用DMEM洗涤细胞两次以除去EDTA,以2×10 5个细胞/ml在含0.1%BSA的DMEM中重悬细胞。
  8. 对于细胞 - 基质粘附测定,将100μl细胞悬浮液(来自步骤7)加入每个胶原蛋白I包被的孔。孵育板在37℃下20分钟,以允许细胞粘附到表面
  9. 向每个孔中加入100μlDMEM以洗掉任何非粘附细胞,洗涤四次 注意:为了实现一致性,请始终使用多通道移液器轻轻添加/移除DMEM多孔井。
  10. 洗涤后,加入含10%FBS的DMEM,并在37℃下孵育细胞4小时以恢复。
  11. 加入10μl的MTT底物到每个孔,并继续在30°C孵育另外2小时。
  12. 接下来,在100μlDMSO(或其他选择的裂解缓冲液)中溶解经MTT处理的细胞,并在分光光度计上测量570nm处的吸光度(参见注释1)。

笔记

  1. 考虑包括以下参考组用于监测该过程的每个步骤:未用胶原I包被的孔; 孔不用DMEM洗涤; 未添加细胞的孔; 孔不添加MTT(MTT测定的背景)。

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Chen等人。 (2009),Humphries等人。 (1998)和Mobley and Shimizu(2001)。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, [参见Chen et al。 (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. Humphries,M。(1998).Curr Protoc Cell Biol 9.1.1-9.1-11。
  3. Mobley,J。和Shimizu,Y。(2001)。 Curr Protoc Immunol 第7章:单元7.28。
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引用:Chen, Y. (2012). Cell Adhesion Assay. Bio-protocol Bio101: e98. DOI: 10.21769/BioProtoc.98;
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Indrani Chakraborty
CSIR-IICB, Kolkata

how to interpret the results of this assay?






4/10/2013 9:49:10 PM Reply
Yanling Chen
Department of Immunology, The Scripps Research Institute, USA

Cell adhesion is a complex process and different factors involved in this process would contribute to the final results. For interpretation of the results, it really depends on what you are looking for and how your experiment is designed. Take just a simple (and ideal) example, if a chemical is capable of directly interfering with cell receptor-ECM interactions, less cell adhering to the surface would be expected to be seen. Most chemicals/proteins may alter cell adhesion by certain indirectly ways, possibly through changes in different cell signaling pathways and remodeling of the cytoskeletal components. Therefore cause must be taken to design and carry out experiments, as well as interpretation of the readouts.

4/15/2013 5:50:26 PM


prashant kurkute
NIPER

Hi yalling ,

I want to do cell adhesion study with my synthesize peptide.
I read your protocol, I am decide to use it , would you please suggest me the paper
from where I get more information on the protocol and how to interpret result ???

Thank you in advance.

6/5/2013 9:33:23 PM


Yanling Chen
Department of Immunology, The Scripps Research Institute, USA

There are actually tons of references for cell adhesion assay. For background information and methods, please see the references(and the references therein) below as examples. I would always recommend to start with some simple tests then make necessary modifications to these protocols.

Liotta et al. Nature2001; 411: 375–9
Fashena et al. Nat Cell Biol2000; 2: E225–9.
Chen etal. Cancer Res2009 69; 3713

7/16/2013 10:36:23 AM