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CBA043 InnoCyte™ Laminin-based 96-Well Cell Invasion Assay

CBA043
  
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      Overview

      Replacement Information

      Key Spec Table

      Detection Methods
      Fluorometric
      Description
      Overview

      This product has been discontinued.



      The InnoCyte™ Laminin-Based 96-well Cell Invasion Assay is designed to determine the invasive capacity of cells using laminin as a significant barrier to invasion or to screen potential anti-metastatic agents in vitro in a convenient, high-throughput, 96-well format. The 8 μm pore size is appropriate for all types of adherent cells such as epithelial, endothelial, or fibroblast cells. The assay is based on the Boyden-chamber principle. Each well of the 96-well cell culture insert contains a polycarbonate membrane with an 8 μm pore size that is coated with a laminin protein layer. The laminin layer forms an effective barrier that prevents non-invasive cells from passing through the 8 μm pores. Invasive cells that can degrade the laminin layer will migrate through the membrane and attach to the underside of the membrane. The invasive cells are dislodged from the underside of the cell culture insert and stained with a fluorescent dye in a single step. Fluorescence is determined using a fluorimeter with an excitation wavelength of ~485 nm and an emission wavelength of ~520 nm.

      Catalogue NumberCBA043
      Brand Family Calbiochem®
      Materials Required but Not Delivered Fluorescence plate reader capable of measuring fluorescence in 96-well plates with a filter set of ~485 nm excitation and ~520 nm emission
      Cells
      Culture medium containing 10% serum
      Serum-free culture medium
      Serum-free culture medium containing chemoattractant(s) (if desired)
      Cell culture incubator equipped with CO2
      Pipettes
      References
      ReferencesCaroll, D.K., et al. 2006. Nat. Cell Biol. 8, 551.
      Ekblom, P., et al. 2003. Matrix Biol. 22, 35.
      Hood, J.D. and Cheresh, D.A. 2002. Nat. Rev. Cancer 2, 91.
      Aumailley, M., et al. 1998. J. Anat. 193, 1.
      Aumailley, M., et al. 1996. J. Invest. Dermatol. 106, 209.
      Burgeson, et al. 1994, Matrix Biol. 14, 209.
      Aznavoorian, S., et al. 1993. Cancer 71, 1368.
      Stetler-Stevenson, W. G., et al. 1993. Annu. Rev. Cell Biol. 9, 541.
      Product Information
      Detection methodFluorometric
      Form96 Tests
      Format96-well plate
      Kit contains96-Well Laminin-Coated 96-Well Cell Invasion Chamber, Black 96-Well Plate, Additional Tray , Calcein-AM Solution, Cell Detachment Buffer, and a user protocol.
      Applications
      Application ReferencesCaroll, D.K., et al. 2006. Nat. Cell Biol. 8, 551.
      Biological Information
      Assay time14-26 h
      Sample TypeCells
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Intended useThe InnoCyte™ Laminin-Based 96-well Cell Invasion Assay is designed to determine the invasive capacity of cells using laminin as a significant barrier to invasion or to screen potential anti-metastatic agents in vitro in a convenient, high-throughput, 96-well format. The 8 µm pore size is appropriate for all types of adherent cells such as epithelial, endothelial, or fibroblast cells.
      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.
      Protect from Light Protect from light
      Avoid freeze/thaw Avoid freeze/thaw
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit contains96-Well Laminin-Coated 96-Well Cell Invasion Chamber, Black 96-Well Plate, Additional Tray , Calcein-AM Solution, Cell Detachment Buffer, and a user protocol.
      Specifications

      Documentation

      InnoCyte™ Laminin-based 96-Well Cell Invasion Assay Certificates of Analysis

      TitleLot Number
      CBA043

      References

      Reference overview
      Caroll, D.K., et al. 2006. Nat. Cell Biol. 8, 551.
      Ekblom, P., et al. 2003. Matrix Biol. 22, 35.
      Hood, J.D. and Cheresh, D.A. 2002. Nat. Rev. Cancer 2, 91.
      Aumailley, M., et al. 1998. J. Anat. 193, 1.
      Aumailley, M., et al. 1996. J. Invest. Dermatol. 106, 209.
      Burgeson, et al. 1994, Matrix Biol. 14, 209.
      Aznavoorian, S., et al. 1993. Cancer 71, 1368.
      Stetler-Stevenson, W. G., et al. 1993. Annu. Rev. Cell Biol. 9, 541.
      User Protocol

      Revision01-June-2009 JSW
      Form96 Tests
      Format96-well plate
      Detection methodFluorometric
      StorageUpon arrival store the Calcein-AM at -20°C and the remaining components at 4°C.
      Intended useThe InnoCyte™ Laminin-Based 96-well Cell Invasion Assay is designed to determine the invasive capacity of cells using laminin as a significant barrier to invasion or to screen potential anti-metastatic agents in vitro in a convenient, high-throughput, 96-well format. The 8 µm pore size is appropriate for all types of adherent cells such as epithelial, endothelial, or fibroblast cells.
      BackgroundTumor invasion and metastasis are the major causes of treatment failure and death in many cancer patients. Invasion is the process whereby tumor cells migrate into the surrounding stroma and disseminate via lymphatic or vascular channels to distant organs. Metastasis is the spread of tumor cells from the primary site to form secondary tumors at other sites in the body. To initiate the metastatic process, neoplastic cells must first penetrate the basement membrane and then invade the interstitial stroma by active proteolysis. Laminins are multi-domain and multifunctional cross-shaped glycoproteins that consist of three distinct, disulfide-linked polypeptide chains (alpha, beta, and gamma). The prototype laminin, laminin-1 (α1-β1-γ1), was isolated from the matrix of the Engelbreth-Holm-Swarm (EHS) tumor. Laminins are structural components of the basement membrane and play a key role in cell adhesion, migration, differentiation and proliferation of several cell types. Basement membranes are the earliest extracellular matrices produced during embryogenesis and provide a support to which epithelial cells can attach, grow and differentiate. They also act as barriers to the passage of cells (with the exceptions of macrophages and lymphocytes) and form a continuous sheet around epithelial structures. The basement membrane is always intact around benign tumors, but malignant tumors show discontinuities in the basement membrane that allow tumor cells to migrate into the surrounding stroma. Laminin is the most abundant structural and biologically active component in basement membranes. Interactions between cancer cells and laminin have been shown to play a critical role in tumor invasion and metastasis. Laminin-1 is the major component of solubilized basement membrane complex from the EHS tumor, a tumor rich in extracellular matrix (ECM) proteins and commonly used in in vitro cell invasion assays. Laminin itself can serve as an effective barrier for non-invasive cells and is employed in various types of cell-based assays. The laminin layer can only be degraded and penetrated by invasive cells and serves as a significant barrier to non-invasive cells.
      Principles of the assayThe InnoCyte™ Laminin-Based 96-Well Invasion Assay is based on the Boyden-chamber principle. Each well of the 96-well cell culture insert contains a polycarbonate membrane with an 8 µm pore size that is coated with a laminin protein layer. The laminin layer forms an effective barrier that prevents non-invasive cells from passing through the 8 µm pores. Invasive cells that can degrade the laminin layer will migrate through the membrane and attach to the underside of the membrane. The invasive cells are dislodged from the underside of the cell culture insert and stained with a fluorescent dye in a single step. Fluorescence is determined using a fluorimeter with an excitation wavelength of ~485 nm and an emission wavelength of ~520 nm.
      Materials provided• 96-well Laminin-Coated Cell Invasion Chamber (Kit Component No. JA9124-1EA): 1 sterile 96-well tray (lower chamber) with laminin-coated cell culture inserts (upper chamber) and lid
      • Black 96-well Plate (Kit Component No. JA7668-1EA): 1 plate supplied as 6 strips, 2 x 8-wells each
      • Additional Tray (Kit Component No. JB306-EA): 1 96-well plastic tray for cell detachment and staining
      • Cell Detachment Buffer (Kit Component No. JA7709-20ML): 1 bottle, 20 ml
      • Calcein-AM Solution (Kit Component No. JA7705-50UL): 2 vials, 50 µl
      Materials Required but not provided Fluorescence plate reader capable of measuring fluorescence in 96-well plates with a filter set of ~485 nm excitation and ~520 nm emission
      Cells
      Culture medium containing 10% serum
      Serum-free culture medium
      Serum-free culture medium containing chemoattractant(s) (if desired)
      Cell culture incubator equipped with CO2
      Pipettes
      Reagent preparationPreparation of Labeling/Cell Detachment Mixture: Prepare just prior to use Note: Preparation of Labeling/Cell Detachment Mixture: Prepare just prior to use as calcein-AM is unstable in aqueous solution and may undergo hydrolysis. Prepare only the volume required for the number of wells to be used in the assay. 1. Warm the Cell Detachment Buffer and Calcein-AM Solution to room temperature. 2. To prepare Labeling/Cell Detachment Mixture for the entire 96-well plate, add 65 µl Calcein-AM Solution to 20 ml Cell Detachment Buffer. 3. Mix well.
      Detailed protocolCell Invasion Assay

      1. Let the 96-Well Laminin-Coated Cell Invasion Chamber adjust to room temperature in a tissue culture hood and warm the serum-free culture medium, and culture medium containing 10% serum to 37°C.
      2. Remove the lid and add 100 µl serum-free medium to the cell culture inserts (upper chamber) to re-hydrate the dried laminin protein layer; replace the lid and incubate at room temp for 15-30 min.
      3. Prepare a cell suspension containing 250,000 - 500,000 cells/ml in serum-free culture medium.
      4. Remove the lid and carefully remove the rehydration solution from step 2 with a pipette without disturbing the laminin-coated membrane. (Note: it does not affect the assay if a small volume of rehydration solution is left in the well)
      5. Lift the 96-well culture insert (upper chamber) or place it upside down without touching the membrane and add 150 µl culture medium containing 10% serum to the wells of the 96-well tray (lower chamber) of the 96-Well Laminin-Coated Cell Invasion Chamber. (Note: if desired chemo-attractants can be used in the place of 10% serum).
      6. Place the cell culture inserts (upper chamber) in the 96-well tray (lower chamber) and add 150 µl of the cell suspension prepared in step 3 to appropriate wells of the cell culture insert (upper chamber); replace the lid.
      7. Incubate for 12-24 h in a CO2 cell culture incubator.

      Dislodging and Labeling of Cells

      1. Add 200 µl of the Labeling/Cell Detachment Mixture to the wells of the Additional Tray supplied with the kit.
      2. Remove the Invasion Chamber from the CO2 cell culture incubator.
      3. Remove the lid and gently remove the 96-well cell culture insert (upper chamber) from the 96-well tray (lower chamber) and carefully flick the medium and cells from the wells. Note: residual cell culture medium or cells in the wells will not affect the assay.
      4. Place the 96-well cell culture insert (upper chamber) in the Additional Tray containing the Labeling/Cell Detachment Mixture. This is the Detachment Chamber Note: The cells that have invaded the laminin layer attach to the underside of the membrane; therefore the bottom surface of the membrane should not be touched or placed on the workspace during the transfer.
      5. Replace the lid and incubate at 37°C for 15-30 min in CO2 tissue culture incubator.
      6. Remove the Detachment Chamber from the CO2 tissue culture incubator and gently tilt the 96-well cell culture insert (upper chamber) back and forth several times to facilitate the cell dislodgement process.
      7. Remove the 96-well cell culture insert (upper chamber), place the lid over the Additional Tray (now containing the dislodged cells), and incubate for an additional 30 min in the CO2 tissue culture incubator.
      8. Remove the Additional Tray from the CO2 tissue culture incubator and transfer 150 µl from each well containing dislodged and labeled cells to the corresponding wells of the Black 96-Well Plate.
      9. Read the fluorescence at ~485 nm excitation and ~520 nm emission.
      Assay characteristics and examples

      Figure 1: Invasive capacity of various cell lines

      HT-1080 (human fibrosarcoma), SK-MEL028 (human melanoma), HeLa (human adenocarcinoma), MCF-7 (human breast adenocarcinoma), and NIH3T3 (murine fibroblast) cells were assessed for invasiveness using the InnoCyte™ Laminin-Based 96-Well Cell Invasion Assay and the Detailed Protocol above. Cells were allowed to migrate/invade the laminin layer in the presence of 10% serum for 18 hr HT-1080 and SK-MEL028 cells exhibited strong invasiveness; HeLa, MCF-7, and NIH3T3 cells did not significantly invade the laminin layer.

      Application referencesCaroll, D.K., et al. 2006. Nat. Cell Biol. 8, 551.
      Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
      InnoCyte™ and Interactive Pathways™ are trademarks of EMD Chemicals, Inc.