QIA129 InnoCyte™ Cell Invasion Assay Kit (24-well)

QIA129
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Key Specifications Table

Detection Methods
Fluorescence

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      Overview

      This product has been discontinued.



      We are offering QCM ECMatrix Cell Invasion Assay, 24-well (8 µm), fluorimetric (Cat. No. ECM554) as a possible alternative. Please read the alternative product documentation carefully and contact technical service if you need additional information.






      A convenient and useful assay for the quantitative measurement of cell invasion. Contains 12 cell culture inserts with an 8 µm polycarbonate membrane. This membrane is coated with a thin layer of a biological matrix. This layer prevents noninvasive cells from going through the membrane. Cells are quantified utilizing a highly sensitive fluorescent dye (Excitation: ~485 nm; Emission: ~520 nm).
      Catalogue NumberQIA129
      Brand Family Calbiochem®
      Materials Required but Not Delivered Pipettors with disposable tips
      96-well plate or strips in black or white color
      Fluorescence plate reader
      Tissue culture equipment
      Forceps
      Serum-free tissue culture medium
      Tissue culture medium with 10% FCS
      References
      ReferencesMontell, D.J. 2003. Nat. Rev. Mol. Cell Biol. 4, 13.
      Egblad, M., et al. 2002. Nat. Rev. Cancer 2, 161.
      Hood, J.D., et al. 2002. Nat. Rev. Cancer 2, 91.
      Chatterjee, N., et al. 2001. Environ. Pathol. Toxicol. Oncol. 20, 211.
      Koul, D., et al. 2001. Oncogene 20, 6669.
      Kleinman, H.K., et al. In Molecular and Cellular Aspects of Basement Membranes, Rohrbach, D.H. and Timpl, R., eds. Academic Press, 1993, pp. 309.
      Repesh, L.A. 1989. Invasion Metastasis 9, 192.
      Albini, A., et al. 1987. Cancer Res. 47, 3229.
      Terranova, V.P., et al. 1986. Proc. Natl. Acad. Sci. USA 83, 465.
      Liotta, L.A., 1984. Am. J. Pathol. 117, 339.
      Product Information
      Detection methodFluorescence
      Form12 Tests
      Format24-well plate
      Kit contains24-Well Plate containing 12 cell culture inserts coated with Basement Membrane Extract, Cell Detachment Solution, Cell Staining Solution, Cell Staining Diluent, and a user protocol.
      Quality LevelMQ100
      Applications
      Biological Information
      Assay time24-48 h
      Sample TypeCulture cells
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Intended useThe Calbiochem® InnoCyte™ Cell Invasion Assay is a quantitative assay for the study of tumor cell invasion in vitro. The assay is useful for discriminating between invasive and non-invasive tumor cell lines and for screening anti-cancer drugs that affect cancer cell invasion. Cell invasion is quantified by labeling the invaded cells with calcein-AM.
      Storage and Shipping Information
      Ship Code Blue Ice Only
      Toxicity Multiple Toxicity Values, refer to MSDS
      Storage Multiple storage requirements
      Storage ConditionsUpon arrival store the Calcein-AM at -20°C and the remaining components at 4°C.
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit contains24-Well Plate containing 12 cell culture inserts coated with Basement Membrane Extract, Cell Detachment Solution, Cell Staining Solution, Cell Staining Diluent, and a user protocol.
      Specifications

      Documentation

      InnoCyte™ Cell Invasion Assay Kit (24-well) SDS

      Title

      Safety Data Sheet (SDS) 

      InnoCyte™ Cell Invasion Assay Kit (24-well) Certificates of Analysis

      TitleLot Number
      QIA129

      References

      Reference overview
      Montell, D.J. 2003. Nat. Rev. Mol. Cell Biol. 4, 13.
      Egblad, M., et al. 2002. Nat. Rev. Cancer 2, 161.
      Hood, J.D., et al. 2002. Nat. Rev. Cancer 2, 91.
      Chatterjee, N., et al. 2001. Environ. Pathol. Toxicol. Oncol. 20, 211.
      Koul, D., et al. 2001. Oncogene 20, 6669.
      Kleinman, H.K., et al. In Molecular and Cellular Aspects of Basement Membranes, Rohrbach, D.H. and Timpl, R., eds. Academic Press, 1993, pp. 309.
      Repesh, L.A. 1989. Invasion Metastasis 9, 192.
      Albini, A., et al. 1987. Cancer Res. 47, 3229.
      Terranova, V.P., et al. 1986. Proc. Natl. Acad. Sci. USA 83, 465.
      Liotta, L.A., 1984. Am. J. Pathol. 117, 339.
      User Protocol

      Revision11-July-2008 JSW
      Form12 Tests
      Format24-well plate
      Detection methodFluorescence
      StorageUpon arrival store the Calcein-AM at -20°C and the remaining components at 4°C.
      Intended useThe Calbiochem® InnoCyte™ Cell Invasion Assay is a quantitative assay for the study of tumor cell invasion in vitro. The assay is useful for discriminating between invasive and non-invasive tumor cell lines and for screening anti-cancer drugs that affect cancer cell invasion. Cell invasion is quantified by labeling the invaded cells with calcein-AM.
      BackgroundThe escape of a tumor mass from confinement by the surrounding capsule or basement membrane signals its progression from a benign to an actively malignant growth state. In the case of epithelial cells, this escape begins with dissolution of the basement membrane that normally underlies the epithelium. The process involves cell adhesion, motility, and the secretion of different classes of proteases. Reconstituted basement membrane matrix is commonly used for the in vitro assessment of cell invasion.
      Principles of the assayThe Calbiochem® InnoCyte™ Cell Invasion Assay utilizes an invasion chamber consisting of a 24-well tissue culture plate and 12 cell culture inserts with an 8-µm pore size polycarbonate membrane. The upper surface of the insert membrane is coated with a uniform layer of dried basement membrane matrix (BMM) solution. The layer of basement membrane solution forms an effective extracellular matrix protein barrier that prevents non-invasive cells from going through the 8-µm pores. Invasive cells are able to degrade the matrix proteins that occlude the pores and pass through and cling to the bottom of the polycarbonate membrane that is tissue culture-treated to enhance cell attachment. Labeling of cells with Calcein-AM and dissociation of the invaded cells from the underside of the membrane are performed in one step. After transfer to a 96-well plate, the samples are measured in a fluorescence plate reader. No tedious manual sample processing, such as removing the biological matrix layer from the topside of the inserts with cotton tip applicators, or cell lysis are required.

      Figure 1: Principle of the assay

      Materials provided• Cell Invasion Chamber (Kit Component No. JA7708-1EA): 1 plate, (lower chamber) 24 wells with 12 cell culture inserts (upper chamber) containing an 8-µm pore size polycarbonate membrane coated with a layer of polymerized and dried basement membrane matrix (BMM) extract
      • Cell Detachment Buffer (Kit Component No. JA7709-15ML): 1 vial, 15 ml
      • Calcein-AM (Kit Component No. JA7705-50UL): 1 bottle, 50 µl
      Materials Required but not provided Pipettors with disposable tips
      96-well plate or strips in black or white color
      Fluorescence plate reader
      Tissue culture equipment
      Forceps
      Serum-free tissue culture medium
      Tissue culture medium with 10% FCS
      Preparation• Cell Suspension: Prepare a cell suspension containing 0.5-1.0 x 106 cells/ml in serum-free medium. If desired, investigational drugs or pharmacological agents that inhibit or stimulate cell invasion can be added directly to the cell suspension prior to adding the cells to the Cell Invasion Chamber.
      Reagent preparation• Cell Staining Solution: Dilute the Calcein-AM 1:300 with Cell Detachment Buffer. For example, to obtain 6 ml Cell Staining Solution (enough to detach cells from 12 cell culture inserts) add 20 µl Calcein-AM to 5980 µl Cell Detachment Buffer. • Standard Curve: If desired, prepare a standard curve for quantitating the number of labeled cells in each sample: 1. Dilute Calcein-AM 1:30 with Cell Detachment Buffer i.e. 20 µl Calcein-AM + 580 µl Cell Detachment Buffer. 2. Add 90 µl of various numbers of cells, prepared in Cell Detachment Buffer, to the wells in a 96-well plate (black or white). 3. Add 10 µl diluted Calcein-AM from step 1 to each well. Include a blank sample without cells. 4. Incubate for 1 h in a CO2 tissue culture incubator and measure fluorescence.
      Detailed protocol1. Warm the Cell Invasion Chamber to room temperature in a tissue culture hood.
      2. Add 300-400 µl warm, serum-free medium to the upper chamber and incubate for 30-60 min at room temperature to rehydrate the BMM extract.
      3. Carefully remove the serum-free medium from step 2 without disturbing the matrix-coated membrane. (Note: it does not affect the assay if a small volume of rehydration solution is left in the compartment).
      4. Add 500 µl medium containing 10% fetal bovine serum to the lower chamber. If desired, chemoattractants can be used in place of serum.
      5. Add 300-350 µl of the cell suspension to the upper chamber.
      6. Incubate for 24-48 h in a CO2 tissue culture incubator.
      7. Add 500 µl Cell Staining Solution to the unused wells of the lower chamber.
      8. Using forceps, gently remove the upper chamber inserts from the lower chamber without touching the underside of the inserts. Discard the cell suspension solution and place the inserts in the wells containing the Cell Staining Solution. Dislodging of the attached cells from the underside can be facilitated by gently tapping the insert (use a forceps) against the bottom of the lower chamber two or three times.
      9. Incubate for 30 min in a CO2 tissue culture incubator.
      10. Following the incubation remove the inserts with forceps and incubate the lower chamber containing the dislodged cells an additional 30 min in a CO2 tissue culture incubator.
      11. Transfer 200 µl of the dislodged cells to duplicate wells of a 96-well plate (black or white).
      12. Measure the fluorescence at an excitation wavelength of 485 ± 10 nm and an emission wavelength of 520 ± 10 nm.
      CalculationsIf desired, results given in relative fluorescence units (RFU) can be converted to cell numbers by running a cell standard curve as illustrated below.

      Figure 2: Quantification of U251 cells

      Increasing numbers of cells were prepared as outlined in the Reagent Preparations section and analyzed for flouresence intensity.

      Example dataOf the four cell lines tested, the human glioma cell line, U251, exhibits the highest invasive capacity, followed by the human fibrosarcoma cell line, HT-1080. The mouse fibroblast cell line, NIH3T3, was used as a negative control. The human cervical cancer cell line, HeLa, is endowed with a very low invasive property, almost non-invasive under the conditions employed. Human gliomas are highly invasive, and remain to be a major obstacle for any effective therapeutic remedy. Among other factors, gliomas express elevated levels of matrix metalloproteases and urokinase. It has been recently reported that HeLa cells bind poorly to vitronectin due to low or no expression of αvβ₃ integrin compared to SiLa cells, a human cervical cancer cell line that expresses high levels of αvβ₃ integrin associated with matrix metalloproteases. Comparative invasion assays demonstrate a much lower invasion potential of HeLa cells than of SiLa cells.

      Figure 3: Assessment of the invasive capacity of various cell lines

      Four cell lines - NIH3T3, U251, HeLa, and HT-1080 - were assessed for invasive properties as outlined in the Detailed Protocol.

      Figure 4: Characteristics

      250,000 U251 (upper row) or NIH3T3 (lower row) cells were added to the upper chamber and incubated overnight at 37°C in a CO2 incubator. The matrix protein layer and cells from the upper chamber were removed before staining the underside of the upper chamber with 0.1% crystal violet to demonstrate movement of the cells through the BMM and membrane.

      Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
      InnoCyte™ is a trademark of EMD Chemicals, Inc.