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Biology

An In vitro FluoroBlok Tumor Invasion Assay

doi: 10.3791/1475 Published: July 20, 2009

Summary

This video demonstrates how to measure cell invasion through cell culture inserts using a fluorescence-based methodology.

Abstract

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to both secrete proteases that break down the basement membrane as well as migrate in order to be invasive. BD BioCoat Tumor Invasion System provides cells with conditions that allow assessment of their invasive property in vitro1,2. It consists of a BD Falcon FluoroBlok 24-Multiwell Insert Plate with an 8.0 micron pore size PET membrane that has been uniformly coated with BD Matrigel Matrix. This uniform layer of BD Matrigel Matrix serves as a reconstituted basement membrane in vitro providing a true barrier to non-invasive cells while presenting an appropriate protein structure to study invasion. The coating process occludes the pores of the membrane, blocking non-invasive cells from migrating through the membrane. In contrast, invasive cells are able to detach themselves from and migrate through the coated membrane. Quantitation of cell invasion can be achieved by either pre- or post-cell invasion labeling with a fluorescent dye such as DiIC12(3) or calcein AM, respectively, and measuring the fluorescence of invading cells. Since the BD FluoroBlok membrane effectively blocks the passage of light from 490-700 nm at >99% efficiency, fluorescently-labeled cells that have not invaded are not detected by a bottom-reading fluorescence plate reader. However, cells that have invaded to the underside of the membrane are no longer shielded from the light source and are detected with the respective plate reader. This video demonstrates an endpoint cell invasion assay, using calcein AM to detect invaded cells.

Protocol

Post-labeling and measurement using BD calcein AM Fluorescent Dye

Cells are labeled for quantitation after they have invaded through the BD Matrigel Matrix and passed through the BD FluoroBlok membrane. As a result, only endpoint measurement of cell invasion may be obtained.

  1. Grow cells to ~80% confluence.
  2. Prepare and rehydrate the insert system.
    1. Remove the package from -20°C storage and allow it to come to room temperature.
    2. Open the foil package and add 500 μL warm (37°C) media to the interior of the insert wells. Allow the plate to rehydrate for 2 hours at 37°C, 5% CO2.
      Note: It is not necessary to rehydrate the uncoated BD Falcon FluoroBlok 24-Multiwell Insert System that will be used as a cell migration control.
    3. After rehydration, carefully remove the medium from the insert wells without disturbing the layer of BD Matrigel Matrix on the membrane. The system is now ready to use.
  3. Prepare cell suspensions by trypsinizing cell monolayers and resuspending the cells in serum-free DMEM at 5 x 104 cells/mL.
    Note: If you are using a different cell type you need to determine the optimal seeding density. To determine the optimal seeding density for your cell type on a porous growth surface, use a range of seeding densities (cells/cm2) that brackets the seeding density used on nonporous surfaces (i.e. flasks, dishes and plates). For example, if you currently seed at 2.5 x 105 cells/cm2, seed at various cell concentrations between 5 x 104 and 5 x 105 cells/cm2 to determine the optimal initial seeding density.
  4. Add 500 μL of cell suspension (2.5 x 104 cells) to the apical chambers.
  5. Add 750 μL of chemoattractant (5% FBS in DMEM) to each of the basal chambers, using the sample ports for access.
  6. Incubate the BD BioCoat Tumor Invasion System and the uncoated BD Falcon FluoroBlok 24-Multiwell Insert Plate for 20-22 hours at 37°C, 5% CO2 atmosphere.
  7. Following incubation, carefully remove medium from the apical chambers. This can be accomplished by flicking the contents into a waste container. Do not touch the bottom surface of the insert system.
  8. Transfer the insert system into a second 24-well plate containing 500 μL/well of 4 μg/mL Calcein AM in HBSS. Incubate for 1 hour at 37°C, 5% CO2. The Calcein AM solution is not removed from the lower chamber before reading fluorescence because it is a nonfluorescent vital dye that is converted into green fluorescent calcein by cytosolic esterases.
  9. Fluorescence of invaded cells is read at wavelengths of 494/517 nm (Ex/Em) on a bottom-reading fluorescent plate reader. The gain setting may need to be determined empirically, but a midpoint gain should be a sufficient starting point. A gain setting that is too high may lead to saturation of the detector with highly fluorescent sample; this may prevent the acquisition of meaningful results. Use of autogain (if supported on your reader) is not recommended. An inverted fluorescence microscope can be used to verify your results; it is especially helpful to do this the first time you run this assay.
    Note: It is of utmost importance that the Insert Systems are read using the correct plate map. For information on loading plate maps see BD Technical Bulletin No. 436 on www.bdbiosciences.com or contact BD Technical Support (labware@bd.com). Proper plate orientation is with well A1 at the top left corner and the BD Flacon logo oriented to the right as the plate is inserted into the reader.

Data Reduction

Data is expressed as in the following equation:
Equation
Background may be subtracted prior to the calculation of percent cell invasion
RFU = relative fluorescent units.

Disclosures

The authors are employees of BD Biosciences that produces reagents and tools used in this article.

Materials

Name Company Catalog Number Comments
BD BioCoat Tumor Invasion System BD Biosciences 354165 or 354166
HT-1080 and NIH/3T3 cells ATCC
Dulbecco’s Modified Eagle Medium (DMEM; serum-free)
Dulbecco’s Phosphate Buffered Saline, without calcium and magnesium (DPBS)
BD Falcon FluoroBlok 24-Multiwell Insert System to be used as a cell migration control BD Biosciences 351157 or 351158
5% Fetal Bovine Serum in DMEM
BD Calcein AM Fluorescent Dye BD Biosciences 354216 or 354217
Hanks’ Balanced Salt Solution (HBSS)
BD Falcon 24-well plates for post cell invasion labeling BD Biosciences 351147
Fluorescence plate reader with bottom reading capabilities PerkinElmer EnVision (R), TECAN Infinite (R), Molecular Devices SpectraMax (R), BioTek Synergy (TM), BMG LABTECH PHERAstar.

DOWNLOAD MATERIALS LIST

References

  1. Sethi, G., Ahn, K. S., Pandey, M. K., Aggarwal, B. B. Celastrol, a novel triterpene, potentiates TNF-induced apoptosis and suppresses invasion of tumor cells by inhibiting NF-κB-regulated gene products and TAK1-mediated NF-ΚB activation. Blood. 109, 2727-2735 (2007).
  2. Takada, Y., Kobayashi, Y., Aggarwal, B. B. Evodiamine Aboloishes Constitutive and Inducible NF-κB Activation by Inhibiting IκBα Kinase Activation, Thereby Suppressing NF-κB-regulated Antiapoptotic and Metastatic Gene Expression, Up-regulating Apoptosis, and Inhibiting Invasion. J. Biol. Chem. 280, 17203-17212 (2005).
  3. Harikumar, K. B., Kunnumakkara, A. B., Ahn, K. S., Anand, P., Krishnan, S., Guha, S., Aggarwal, B. B. Modification of the cysteini residues in IκBα kinase and NF-κB (p65) by xanthohumol leads to suppression of NF-κB-regulated gene products and potentiation of apoptosis in leukemia cells. Blood. 113-2003 (2009).
  4. Nair, A. S., Sishodia, S., Ahn, K. S., Kunnumakkara, A. B., Sethi, G., Aggarwal, B. B. D. eguelin an Akt Inhibitor, Suppresses IΚBα Kinase Activation Leading to Suppression of NF-κB-Regulated Gene Expression, Potentiation of Apoptosis, and Inhibition of Cellular Invasion. J. Immunol. 177-5612 (2006).
  5. Partridge, J., Qian, S. Quantitative and High-Throughput Screening of Tumor Cell Invasion using a Cell-based Model. Society for Biomolecular Screening 2005 Conference. 2005 Sept 11-15, Geneva, Switzerland, (2005).
  6. An Improved In Vitro Device For Measuring Cell Invasion and Method for its Formation. US patent. Mannuzza, F., Flaherty, P., Ilsley, S., Kramer, M. 6,740,501,B2 (2004).
  7. Flaherty, P., Goldberger, A., Dery, O. Effect of Fluorescent Cell Labeling on Tumor Invasion and Screening of Anti-Metastatic Compounds. Mole. Biol. Cell. 12, 46a-46a (2001).
  8. Flaherty, P., Mannuzza, F., Ilsley, S., Maliakal, J., Wu, M. Screening of Anti-Metastatic Compounds by a Fluorescence Based Tumor Invasion Assay. Screentech 2001 Conference. 2005 Mar 12-16, San Diego, CA, (2005).
  9. Mannuzza, F., Flaherty, P., Wu, M., Ilsley, S. Development of a Quantitative, High-Throughput Assay System for the Discovery of Anti-Cancer Drugs. Proceedings of the Society for Biomolecular Screening. 1999 Sept 13-16, Edinburgh, SC, UK, (1999).
An <em>In vitro</em> FluoroBlok Tumor Invasion Assay
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Cite this Article

Partridge, J., Flaherty, P. An In vitro FluoroBlok Tumor Invasion Assay. J. Vis. Exp. (29), e1475, doi:10.3791/1475 (2009).More

Partridge, J., Flaherty, P. An In vitro FluoroBlok Tumor Invasion Assay. J. Vis. Exp. (29), e1475, doi:10.3791/1475 (2009).

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