Name: An In vitro FluoroBlok Tumor Invasion Assay (Video) | JoVE Journal
Uploaded: 2009-07-20T13:00:00.000+00:00
Duration: 7 min 53 s
Description: Learn a fluorescence-based tumor invasion assay to measure cancer cell migration through basement membrane using HT 1080 and NIH 3T3 cell lines. This method provides reliable quantification of invasive behavior.

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5 minutes

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.
<ol>
<li>Grow cells to ~80% confluence.</li>
<li>Prepare and rehydrate the insert system.<ol style="list-style-type:lower-alpha;">
<li>Remove the package from -20°C storage and allow it to come to room temperature.</li>
<li>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.</li>
<li>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.</li>
</ol></li>
<li>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.</li>
<li>Add 500 μL of cell suspension (2.5 x 104 cells) to the apical chambers.</li>
<li>Add 750 μL of chemoattractant (5% FBS in DMEM) to each of the basal chambers, using the sample ports for access.</li>
<li>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.</li>
<li>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.</li>
<li>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.</li>
<li>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.</li>
</ol>
Data Reduction
Data is expressed as in the following equation: <img src="/files/ftp_upload/1475/1475eqa.jpg" alt="Cell invasion calculation; equation for % invasion using RFU values, migration, chemotaxis analysis." data-altupdatedat="1787630408000" data-alt="Equation "> Background may be subtracted prior to the calculation of percent cell invasion RFU = relative fluorescent units.

<ol><li>Sethi, G., Ahn, K. S., Pandey, M. K., Aggarwal, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Celastrol%2C+a+novel+triterpene%2C+potentiates+TNF-induced+apoptosis+and+suppresses+invasion+of+tumor+cells+by+inhibiting+NF-%CE%BAB-regulated+gene+products+and+TAK1-mediated+NF-%CE%9AB+activation%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">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.</a> Blood. 109, 2727-2735 (2007).</li>
<li>Takada, Y., Kobayashi, Y., Aggarwal, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evodiamine+Aboloishes+Constitutive+and+Inducible+NF-%CE%BAB+Activation+by+Inhibiting+I%CE%BAB%CE%B1+Kinase+Activation%2C+Thereby+Suppressing+NF-%CE%BAB-regulated+Antiapoptotic+and+Metastatic+Gene+Expression%2C+Up-regulating+Apoptosis%2C+and+Inhibiting+Invasion%5BTitle%5D)+AND+%22J.+Biol.+Chem%22%5BJournal%5D)">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.</a> J. Biol. Chem. 280, 17203-17212 (2005).</li>
<li>Harikumar, K. B., Kunnumakkara, A. B., Ahn, K. S., Anand, P., Krishnan, S., Guha, S., Aggarwal, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modification+of+the+cysteini+residues+in+I%CE%BAB%CE%B1+kinase+and+NF-%CE%BAB+%28p65%29+by+xanthohumol+leads+to+suppression+of+NF-%CE%BAB-regulated+gene+products+and+potentiation+of+apoptosis+in+leukemia+cells%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">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.</a> Blood. , 113-2003 (2009).</li>
<li>Nair, A. S., Sishodia, S., Ahn, K. S., Kunnumakkara, A. B., Sethi, G., Aggarwal, B. B. D. eguelin <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((an+Akt+Inhibitor%2C+Suppresses+I%CE%9AB%CE%B1+Kinase+Activation+Leading+to+Suppression+of+NF-%CE%BAB-Regulated+Gene+Expression%2C+Potentiation+of+Apoptosis%2C+and+Inhibition+of+Cellular+Invasion%5BTitle%5D)+AND+%22J.+Immunol%22%5BJournal%5D)">an Akt Inhibitor, Suppresses IΚBα Kinase Activation Leading to Suppression of NF-κB-Regulated Gene Expression, Potentiation of Apoptosis, and Inhibition of Cellular Invasion.</a> J. Immunol. , 177-5612 (2006).</li>
<li>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).</li>
<li>An Improved In Vitro Device For Measuring Cell Invasion and Method for its Formation. US patent. , 6,740,501,B2 (2004).</li>
<li>Flaherty, P., Goldberger, A., Dery, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+Fluorescent+Cell+Labeling+on+Tumor+Invasion+and+Screening+of+Anti-Metastatic+Compounds%5BTitle%5D)+AND+%22Mole.+Biol.+Cell%22%5BJournal%5D)">Effect of Fluorescent Cell Labeling on Tumor Invasion and Screening of Anti-Metastatic Compounds.</a> Mole. Biol. Cell. 12, 46a-46a (2001).</li>
<li>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).</li>
<li>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).</li>
</ol>

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

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

an in vitro fluoroblok tumor invasion assay

An In vitro FluoroBlok Tumor Invasion Assay

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 &gt;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.

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

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 ...

Cell invasion is an important step in cancer progression and angiogenesis. In vitro invasion assays typically assess the ability of cells to break down reconstituted basement membrane, such as BD matrix, gel matrix, and migrate through cell culture inserts. The tumor invasion assay begins when cells are added to Fluor block inserts coated with matri gel.A reconstituted basement membrane chemo attractant is added to the basal chamber and the system is incubated. Overnight invasive cells will be able to degrade the matri gel barrier and pass through the flora block membrane, a specialized membrane that protects Fluor from ambient light. The contents of the apical chamber are discarded and the multi-well insert is added to a second plate containing a Fluor.After incubation, viable cells are stained and can be detected both visually and by a microplate reader. Hi, I'm Jeff Partridge from BD Biosciences Discovery LabWare. Today we'll show you a procedure for floor block tumor invasion assay.We use this procedure to study the invasion of tumor cells through a basement membrane towards the chemo attractant. So let's get started. Grow the cells for the tumor invasion assay to about 80%confluence.In this experiment, we will be using HT 10 80 and N NIH three T three cell lines. HT 10 80 cells are proven to migrate and invade the matrigel barrier in the system and hence serve as good positive controls for the assay. NIH three T three cells are not invasive, but do migrate and hence serve as good negative controls for this procedure.To begin, the assay rehydrate the system by removing the BD bio coat tumor invasion system Package from minus 20 degrees Celsius storage and allow it to come to room temperature. Since the assay requires sterile technique, let the insert come to room temperature in the tissue culture hood. Once the packages of room temperature, open it to rehydrate the basement membrane through which your cells of interest may pass.At 500 microliters of warm medium to the interior of the insert wells, allow the plate to rehydrate for two hours at 37 degrees Celsius, 5%carbon dioxide. There is no need to rehydrate the uncoated BD Falcon Flora Block 24 multi-well insert plate as it will be used as a cell migration control to prepare the cells for the invasion assay. Trypsin is the cell mono layers and resus suspend the cells in serum free DMEM at five times 10 to the four cells per milliliter.If a different cell type is used, the optimal seeding density must be determined empirically. After rehydration of the coated plate, carefully remove the medium from the insert wells without disturbing the layer of MA gel on the membrane. The system is now ready to be used.Do not let the MA gel layer dry out. To begin the cell invasion assay, add 500 microliters of each cell suspension to the apical chambers of the tumor invasion system in both the matrigel coated and uncoated configurations. Next, using the sample ports for access, add 750 microliters of chemo attractant to the basal chambers within both coated and uncoated plates.It is extremely important not to introduce any bubbles while filling the basal chambers. As a migration control, add each cell suspension as well as the chemo attractant to the uncoated BD Falcon Flora Block 24 Multi WA insert plate. Incubate the Tumor invasion System and the uncoated Falcon Flora Block 24 Multi WA insert plate for 20 to 22 hours at 37 degrees Celsius, 5%carbon dioxide atmosphere on the following day.When the incubation is complete, the cells are ready to be labeled with a fluorescent dye calcium am. Carefully remove the medium from the apical chambers by flicking the contents into a waste container. Then immediately transfer the insert system into a second 24 well plate containing 500 microliters per well of four micrograms per milliliter.Calcium am in Hank's balanced salt solution. Repeat these steps with migration control. Incubate the plates for one hour at 37 degrees Celsius, 5%carbon dioxide.Do not touch the bottom surface of the insert system. At the end of the one hour incubation with calcium am the fluorescence of the invading cells can be measured. It is of utmost importance that the insert systems are red using the correct plate map.Proper plate orientation is with well A one at the top left corner and the BD falcon logo oriented to the right as the plate is inserted into the reader fluorescence is read at wavelengths of 494 517 nanometers on a bottom reading fluorescent plate reader. You should verify your results with an inverted fluorescence microscope to ensure the plate reader data matches the visual results. These are typical plate reader data from a coated plate and an uncoated control plate obtained from cell invasion assays of the two cell lines HT 10 80 and NIH three T three.Data from wells with no cells represent the background that may be subtracted prior to the calculation of percent cell invasion. Calculate percent invasion use the following equation. Percent invasion equals the mean relative fluorescent units or rfu of cells that have passed through the matrigel coated membrane towards the chemo attractant.This is divided by the mean RFU of cells that have migrated through the uncoated VD flua block membrane towards the chemo attractant. This value is then multiplied by 100. This graph shows comparison of the percent cell invasion between HT 10 80 and NIH three T three cells, and as you can see, HT 10 80 cells are invasive while NIH three T three cells are not.When the cells are checked under an inverted fluorescent microscope, the results are consistent with those obtained from the plate reader. The invasive HT 10 80 cells invaded through the matrigel as well as migrated through the uncoated membrane. In contrast, the non-invasive NIH three T three cells did not invade through the matrigel, but migrated through the uncoated membrane.We've just shown you how to perform a floral block tumor invasion assay. When doing this procedure, it's important to keep the plates horizontal and minimize formation of bubbles. These could affect your results.So that's it. Thanks for watching and good luck with your experiments.

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Learn a fluorescence-based tumor invasion assay to measure cancer cell migration through basement membrane using HT 1080 and NIH 3T3 cell lines. This method provides reliable quantification of invasive behavior.

An In vitro FluoroBlok Tumor Invasion Assay

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