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 JoVE Biology

An In vitro FluoroBlok Tumor Invasion Assay

1, 1

1BD Biosciences, Discovery Labware

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    Summary

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

    Date Published: 7/20/2009, Issue 29; doi: 10.3791/1475

    Cite this Article

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

    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.

    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. 113:2003-2013 (2009).
    4. Nair, A.S., Sishodia S., Ahn, K.S., Kunnumakkara, A.B., Sethi, G., Aggarwal, B.B. Deguelin, 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. 177:5612-5622 (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. Geneva, Switzerland (http://www.bdbiosciences.com/discovery_labware/Products/pdf/S05B146.pdf)
    6. US 6,740,501,B2 , 2004 "An Improved In Vitro Device For Measuring Cell Invasion and Method for its Formation" Mannuzza, F., Flaherty, P., Ilsley, S., Kramer, M.
    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:S 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, San Diego, CA (2001).
    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, Edinburgh, SC,UK (1999).

    Comments

    22 Comments

    Hy, is possible to use this system with cells GFP positive, if yes how is the method.

    thanks adriana
    Reply

    Posted by: AnonymousJune 4, 2010, 4:45 AM

    Adriana,

    This can be done with very few changes from the above protocol.

    Use the same cell density specified, but instead of staining after step 7, leave the medium in the apical chambers. (Do not take apart the system.) Simply read the plate as normal; GFP and calcein AM have similar spectra.

    You can gather kinetic data from time zero in this manner, if you wish.

    A useful reference is the specific product's guidelines for use, available on our website.

    I hope this helps.

    Jeff Partridge
    BD Biosciences
    Reply

    Posted by: Jeff P.June 9, 2010, 11:50 AM

    hello , I want to know how many cell lines can be performed in BD matrigel invasion assay chamber of ²4 well plate . As i am working in 5 carcinoma cell line including the HT1080 . kindly help me how to perform this assay i have gone through the protocol and the vedio am doing for the first time . thank you
    Reply

    Posted by: stella mary r.July 19, 2010, 6:23 AM

    Thank you for watching.

    If you are referring to cell lines in general, I would advise you to try any lines you think may be invasive.
    If you mean can you try five different cell lines on one insert system (plate), you could easily do that with n=3 (replicates per cell line), and still get useful data.

    I hope this helps.

    Jeff Partridge
    BD Biosciences
    Reply

    Posted by: Jeff P.July 20, 2010, 1:18 PM

    Thank you Jeff patridge

    Regards
    stella
    Reply

    Posted by: stella mary r.July 21, 2010, 5:05 AM

    Hello jeff

    After the staining procedure in matrigel invasion chamber assay .How to identify the invaded cells and migrated cells as in control and test .After staining the cells are stained as pale red and red colur want to know how to count them.

    Thanks
    Reply

    Posted by: stella mary r.July 23, 2010, 4:17 AM

    Stella,

    Please see our website for information about the Matrigel Invasion Chamber. The focus of this article is FluoroBlok.
    In general, the systems are similar, but there are some differences that I cannot detail here.

    Jeff Partridge
    BD Biosciences
    Reply

    Posted by: Jeff P.July 27, 2010, 1:25 PM

    Hello Jeff
    As am doing the manual matrigel invasion chamber assay not the fluoroblok one, so i want to know is the cell count will be same as that u g et in fluoroblok . Please help in this issue.

    Thanks stella
    Reply

    Posted by: stella mary r.July 26, 2010, 4:41 AM

    Stella,

    The trends will be similar, but the measurement systems are different: clear inserts are measured by visual counting of cells while FluoroBlok is measured using fluorescence detection.

    Jeff Partridge
    BD Biosciences
    Reply

    Posted by: Jeff P.July 27, 2010, 1:28 PM

    I would like to perform a similar assay using neutrophils. This is to demonstrate anti-inflammation activity of some of my plant extracts. Is the matrigel necessary for my type of experiments? Or do you have inserts without the matrigel
    Reply

    Posted by: AnonymousOctober 20, 2010, 4:02 AM

    Ronald,

    We have two technical bulletins that specifically reference immune cells and inserts:

    Technical Bulletin #407 - An In Vitro Assay for Study of Neutrophil Migration Through Interstitial Matrix Using BD Falcon&#x²1²²; Cell Culture Inserts

    Technical Bulletin #457 - Optimized Chemotaxis Conditions for Primary Blood Monocytes or THP-1 Cells Using BD Falcon&#x²1²²; FluoroBlok&#x²1²²; 96-Multiwell Insert Plates

    These can be found in the Cell Culture Resources section of the BD Biosciences website at:
    www.bdbiosciences.com/support/resources/cellculture/index.jsp

    Jeff Partridge
    BD Biosciences
    Reply

    Posted by: Jeff P.October 20, 2010, 11:01 AM

    Hi Jeff,

    I am wondering whether it is possible to isolate those invaded tumor cells for genomic DNA extraction. If yes, it there a protocol available?

    Thanks,
    Yukun
    Reply

    Posted by: AnonymousOctober 28, 2010, 5:40 PM

    Yukun,

    You should be able to use enzymatic means to remove cells from the undersides of the inserts, similar to removing cells from TC surfaces.

    Jeff Partridge
    BD Biosciences
    Reply

    Posted by: Jeff P.November 2, 2010, 9:06 AM

    Our microplate reader is not inside of our cell culture room. Due to safety restrictions, we cannon take cells out of our room. Is it possible to add Formaldehyde 4% during the post-labeling step without affecting/compromising the membranes? I know formaldehyde is used to do fluorescence readings, but we want to make sure we wont damage the results by adding them into the Calcein solution.
    Reply

    Posted by: AnonymousNovember 3, 2010, 3:11 PM

    Hi,
    I am interested in doing this assay in 96-well plates. The procedure to be followed would be same except for the amount of volume the wells can hold. So the cell suspension volume, chemoattractant and calcien volume to be prepared and aliquoted to indiviual wells may vary from that of ²4-well plates. Can you please give the dimensions of 96-well plates, apical chamber.
    Thanks
    Reply

    Posted by: AnonymousJanuary 15, 2012, 9:03 PM

    Reji,

    The quick answer is 3.18 mm diameter, but we do sell a 96 multiwell version of the tumor invasion system. Those guidelines for use are found at:
    http://www.bdbiosciences.com/external_files/dl/doc/manuals/live/web_enabled/354167_354168_pug.pdf
    while set-up guidelines (²4 well and 96 well) for selected plate readers are at:
    http://www.bdbiosciences.com/external_files/dl/doc/tech_bulletin/live/web_enabled/tb436.pdf
    You can always find more information at:
    http://www.bdbiosciences.com
    Thanks,
    Jeff
    Reply

    Posted by: Jeff P.January 16, 2012, 3:45 PM

    Hi,
    Thanks for the email. I have got the cell invasion kit from BD and about to start my experiment. I was looking into the link that you had sent me for the 96-well plate.
    1. However, I have got some doubt pertaining to the testing of my novel anti-cancer compounds.
    The intention of the experiment is to test the inhibitory potency of cell invasion through HT-1080 cells. When (at what stage) should I introduce the inhibitory compound solution into the apical chamber? (It²17;s not mentioned in the protocol).
    ². On the instrument settings:
    I will be measuring the end-point (NOT- real time)
    Sensitivity
    If this is going to be a low-signal endpoint assay (I am not sure what that means), 100 readings per well may be optimal as per the instrument manual. BUT,
    In fluorescence intensity reading, the sample may photo bleach. SO it is recommended to use 10 readings or less. WHAT NUMBER OF READING WOULD BE OPTIMAL FOR THIS ASSAY?

    Setting of gain-intensity, which would be optimal, HIGH or LOW.

    In the technical bulletein #436, Set up guidelines are not mentioned for FLEX STATION 3 with SoftMax Pro 5, on which I would be doing my measurement. Your advice and suggestion would be very helpful as the instrument manufacture technical support group has nothing to contribute to this.

    Thanks,
    Reji
    Reply

    Posted by: AnonymousFebruary 27, 2012, 8:35 PM

    Reji,

    please contact BD Biosciences Discovery Labware technical support at 877.²3².8995.

    Jeff
    Reply

    Posted by: Jeff P.March 5, 2012, 1:01 PM

    Hi,
    I was wondering if I could use this system to meassure oxitaxis. any suggestions?

    Thank you
    Reply

    Posted by: Ana C.September 25, 2012, 5:00 PM

    Ana,

    if you mean migration in an oxygen gradient, I do not think that could easily be done using this system.

    Jeff
    Reply

    Posted by: Jeff P.September 26, 2012, 8:29 AM

    Hola , la duda que tengo es de donde se obtienen los datos de:

    MEAN RFU OF CELL MIGRATED THROUGH UNCOATED BD FLUOROBLOK MEMBRANE TOWARDS CHEMOATTRACTANT

    gracias
    para hacer el c&#²²5;lculo del % de invasi&#²43;n
    Reply

    Posted by: rocio m.October 24, 2012, 2:31 PM

    Rocio-

    this is part of the calculation of the % invasion (invasion index). We compare invasion through Matrigel then effectively use migration without Matrigel as the baseline.

    Jeff
    Reply

    Posted by: Jeff P.October 25, 2012, 10:38 AM

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