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Cell Adhesion Assay   

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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.
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Chen, Y. (2012). Cell Adhesion Assay. Bio-101: e98. DOI: 10.21769/BioProtoc.98.
Q&A
By submitting a question/comment you agree to abide by our Terms of Service. If you find something abusive or that does not comply with our terms please contact us at eb@bio-protocol.org.

If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.

If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.

ÖZGE DENİZ YEŞİL
yeditepe universtiy
do you have any tutorial video for cell adhesion assay experiment?
4/4/2020 2:04:03 PM Reply
Indrani Chakraborty
CSIR-IICB, Kolkata

how to interpret the results of this assay?






4/10/2013 9:49:10 PM Reply
Yanling Chen
The Scripps Research Institute

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 Reply


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 Reply


Yanling Chen
The Scripps Research Institute

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 Reply


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