CBA017 InnoCyte™ Cell Migration Assay, 24-well plate

CBA017
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      Overview

      Replacement Information

      Key Specifications Table

      Detection Methods
      Fluorometric

      Pricing & Availability

      Catalog Number AvailabilityPackaging Qty/Pack Price Quantity
      CBA017-1KIT
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          Description
          OverviewA convenient assay for chemotaxis cell migration in 24-well plates. Cells migrate through a 8 μm pore to a feeder layer containing serum or other attractant. Migrated cells are quantitated using a cell-permeable fluorescent dye.
          Catalogue NumberCBA017
          Brand Family Calbiochem®
          Materials Required but Not Delivered 1X PBS (sterile)
          Cell culture medium (serum-free)
          Pipettors with disposable tips
          Fluorescence reader capable of measuring fluorescence in 96-well plates with a filter set of ~485 nm (excitation) and 520 nm (emission).
          Hemocytometer and microscope for cell counting
          Tissue culture facility
          Forceps
          References
          ReferencesWells, A., et al. 2002. Acta Oncol. 41, 124.
          Staff, A. C. 2001. Scand. J. Clin. Lab. Invest. 61, 257.
          Eatock, M.M., et al. 2000. Cancer Treat. Rev. 26, 19.
          Klinghoffer, R.A., et al. 1999. EMBO J. 18, 2459.
          Sieg, D.J., et al. 1999. J. Cell Sci. 112, 2677.
          Smilenov, L. B., et al. 1999. Science 286, 1172.
          Burridge, K. et al. 1997. Trends Cell Biol. 7, 342.
          Huttenlocher, A., et al. 1997. J. Biol. Chem. 272, 32719.
          Price, J.T., et al. 1997. Crit. Rev. Biochem. Mol. Biol. 32, 175.
          Lauffenburger, D.A., and Horowitz, A.F. 1996. Cell 84, 359.
          Product Information
          Detection methodFluorometric
          Form12 Tests
          Format24-well plate
          Kit contains12 Sterile Cell Culture Inserts, Sterile 24-well Plate, Black Plate Strips, Cell Detachment Buffer, Fluorescent Dye, Latrunculin A (inhibitor), and a user protocol.
          Applications
          Biological Information
          Assay time3-5 h
          Sample TypeAdherent cells
          Physicochemical Information
          Emission max.
          Excitation max.
          Dimensions
          Materials Information
          Toxicological Information
          Safety Information according to GHS
          Safety Information
          Product Usage Statements
          Intended useThe InnoCyte™ Cell Migration Assay, 24-Well is suited for studying the effects of various drugs or agents on cell motility and for identifying chemoattractant agents. The Cell Culture Inserts have an 8-µm pore size membrane that is suitable for migration of epithelial, mesenchymal, and endothelial cell types. The membrane is not coated with an extracellular matrix protein, so is not suitable for haptotactic migration studies. The assay is not intended for leukocyte migration experiments; leucocytes require membranes with a pore size that is smaller than 8 µm.
          Storage and Shipping Information
          Ship Code Blue Ice Only
          Toxicity Multiple Toxicity Values, refer to MSDS
          Storage +2°C to +8°C
          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 contains12 Sterile Cell Culture Inserts, Sterile 24-well Plate, Black Plate Strips, Cell Detachment Buffer, Fluorescent Dye, Latrunculin A (inhibitor), and a user protocol.
          Specifications

          Documentation

          InnoCyte™ Cell Migration Assay, 24-well plate SDS

          Title

          Safety Data Sheet (SDS) 

          InnoCyte™ Cell Migration Assay, 24-well plate Certificates of Analysis

          TitleLot Number
          CBA017

          References

          Reference overview
          Wells, A., et al. 2002. Acta Oncol. 41, 124.
          Staff, A. C. 2001. Scand. J. Clin. Lab. Invest. 61, 257.
          Eatock, M.M., et al. 2000. Cancer Treat. Rev. 26, 19.
          Klinghoffer, R.A., et al. 1999. EMBO J. 18, 2459.
          Sieg, D.J., et al. 1999. J. Cell Sci. 112, 2677.
          Smilenov, L. B., et al. 1999. Science 286, 1172.
          Burridge, K. et al. 1997. Trends Cell Biol. 7, 342.
          Huttenlocher, A., et al. 1997. J. Biol. Chem. 272, 32719.
          Price, J.T., et al. 1997. Crit. Rev. Biochem. Mol. Biol. 32, 175.
          Lauffenburger, D.A., and Horowitz, A.F. 1996. Cell 84, 359.
          User Protocol

          Revision08-July-2014 JSW
          Form12 Tests
          Format24-well plate
          Detection methodFluorometric
          StorageUpon arrival store the Calcein-AM at -20°C and the remaining components at 4°C.
          Intended useThe InnoCyte™ Cell Migration Assay, 24-Well is suited for studying the effects of various drugs or agents on cell motility and for identifying chemoattractant agents. The Cell Culture Inserts have an 8-µm pore size membrane that is suitable for migration of epithelial, mesenchymal, and endothelial cell types. The membrane is not coated with an extracellular matrix protein, so is not suitable for haptotactic migration studies. The assay is not intended for leukocyte migration experiments; leucocytes require membranes with a pore size that is smaller than 8 µm.
          BackgroundCell migration is crucial for embryonic development inflammatory immune responses, wound repair, and tumor formation and metastasis. Recent findings highlight the importance of tension and the actin and myosin filament network, microtubules, and the Rho/Rac family of signaling molecules in the process of cell migration. Migratory cells must coordinate these complex processes. The regulation of focal adhesion and focal complex turnover is critical for the continued remodeling and reorganization of adhesion contacts during cell migration. Migratory defects have been reported in cells lacking Src family kinases, focal adhesion kinase (FAK), and calpain. The cellular changes required for malignant conversion are also complex. A common denominator among these cellular changes is aberrant regulation of cell migration. Examples of these changes include secretion of proteases, decreased synthesis of protease inhibitors, loss of cell-to-cell contact, and alterations in the response to and production of chemotactic stimuli during tumor-induced angiogenesis and tumor invasion. Endothelial cell motility and related aspects of the neo-vascularization process, such as cell adhesion and extracellular matrix remodeling, are promising targets for anti-angiogenesis drug development. Tumor cell invasion and metastasis, which represent later points in cancer progression, also inherently involve cell motility.
          Principles of the assayThe Innocyte™ Cell Migration Assay, 24-Well uses a Cell Culture Insert with an 8-µm pore size membrane as the migration chamber. Cells are harvested, washed, resuspended in serum-free medium, and applied to the Cell Culture Insert. Cell culture medium containing serum and/or chemotactic agents is applied to the well(s) of the 24-Well Cell Culture Plate and the Cell Culture Inserts containing cells are placed in the wells. Cell migration through the membrane is assessed by staining the cells that attach to the lower side of the membrane with Calcein-AM, a fluorescent dye. Staining and dislodgement of the cells from the lower side of the membrane is performed simultaneously; no manual cell scraping or cell counting is required. The Calcein-AM is a cell-permeable dye that is cleaved by intracellular esterases, resulting in fluorescence. The samples are transferred to the Black Module Strips and fluorescence is measured at 485 nm (excitation) and 520 nm (emission). Latrunculin A Solution (an anti-migratory compound) is supplied as an optional negative control.

          Figure 1: Assay Principle

          Materials provided• Sterile 24-Well Cell Culture Plate (Kit Component No. JB111-1EA): 1 plate
          • Cell Culture Inserts (Kit Component No. JA7900-1EA): 12 cell culture inserts with an 8 µm pore size, individually sterile-packaged
          • Black Module Strips (Kit Component No. JA7901-EA): supplied as 2 16-well strips in a frame
          • Cell Detachment Buffer (Kit Component No. JA7709-8ML): 1 bottle, 8 ml
          • Calcein-AM (Kit Component No. JA7705-50UL): 1 amber vial, 50 µl,
          • Latrunculin A Solution (Antimigratory Compound) (Kit Component No. JA7832-25UL): 1 amber vial, 25 µl, supplied as 1 mM
          Materials Required but not provided 1X PBS (sterile)
          Cell culture medium (serum-free)
          Pipettors with disposable tips
          Fluorescence reader capable of measuring fluorescence in 96-well plates with a filter set of ~485 nm (excitation) and 520 nm (emission).
          Hemocytometer and microscope for cell counting
          Tissue culture facility
          Forceps
          Reagent preparation• Cell Detachment Buffer: Warm the Cell Detachment Buffer and Calcein-AM to room temperature. Prepare 0.5 ml/well cell labeling/detachment solution by diluting the Calcein-AM 1:300 in Cell Detachment Buffer (i.e. 10 µl Calcein-AM in 3 ml Cell Detachment Buffer).
          Detailed protocol1. Remove the Cell Culture Inserts from the package and place them in the upper two rows of the Sterile 24-Well Cell Culture Plate (if not all inserts are used at once, additional 24-well tissue culture plate(s) are required (not provided) for future experiments. The assembled plate is now referred to as the migration chamber; the Cell Culture Insert is the upper chamber and the Sterile 24-Well Cell Culture Plate is the lower chamber.
          2. Harvest cells by method of choice.
          3. Wash cells 1-2 times with 1X PBS to remove residual culture medium.
          4. Resuspend the cell pellet in serum-free cell culture medium to yield a final cell concentration of approximately 4-8 x 105 cells/ml.
          5. Add 350 µl cell suspension to each Cell Culture Insert (upper chamber). If desired, compounds affecting cell motility may be added at the appropriate concentration to the cell suspension prior to loading the cells in the Cell Culture Insert. It is recommended that the cell suspension be incubated with desired compounds for 10-15 min at 37°C in a CO2 tissue culture incubator prior to adding the cell suspension to the Cell Culture Insert. If desired a negative control can be prepared by adding Latrunculin A Solution to a separate sample (350 µl) of cell suspension to a final concentration of 1-3 µM; pre-incubate as recommended above.
          6. Add 500 µl serum-free cell culture medium containing chemoattractant of choice to desired number of wells in the Sterile 24-Well Cell Culture Plate (lower chamber) by carefully pipetting between the walls of the upper and lower chambers; alternatively cell culture medium containing serum may be added to the desired number of wells in the 24-well Cell Culture Plate. For the negative control use only serum-free cell culture medium.
          7. Place the lid on top of the migration chamber and incubate 3-24 h at 37°C in a CO2 tissue culture incubator.
          8. Remove the migration chamber from the tissue culture incubator and add 0.5 ml of the above cell labeling/detachment solution in the unused rows of the 24-well Cell Culture Plate.
          9. Remove Cell Culture Inserts with forceps and carefully discard remaining cells and cell culture medium. Note: migratory cells attach to the bottom of the Cell Culture Insert membrane, hence the bottom surface of the membrane should not be disturbed.
          10. Place the Cell Culture Inserts in the wells containing the cell labeling/detachment solution.
          11. Place the lid on the Sterile 24-Well Cell Culture Plate and incubate 20 min at 37°C in a CO2 tissue culture incubator.
          12. Remove the Cell Culture Inserts from the Sterile 24-Well Cell Culture Plate. Using forceps gently tap each Cell Culture Insert against the bottom of the well to ensure complete removal of cells. Incubate the Sterile 24-Well Cell Culture Plate containing the dislodged cells for an additional 40 min at 37°C in a CO2 tissue culture incubator.
          13. Remove the Sterile 24-Well Cell Culture Plate from the CO2 tissue culture incubator and transfer 200 µl of the dislodged and labeled cells, in duplicate, to an appropriate number of wells on the Black Module Strips.
          14. Measure fluorescence with a fluorescence plate reader 485 nm (excitation) and 520 nm (emission).
          Assay characteristics and examples

          Figure 2: HT-1080 cell Migration

          HT-1080 cells were allowed incubated in the presence or absence of serum and Latrunculin A Solution for 3 h at 37°C in a 6% CO2 tissue culture incubator (chemotactic migration) as described in the Detailed Protocol. Approximately 150,000 cells were loaded into triplicate Cell Culture Inserts. Migratory cells were subsequently dislodged and labeled as described in the Detailed Protocol.


          Figure 3: HeLa Cell Migration

          HeLa cells were incubated in the presence or absence of serum for 3 and 20 h at 37°C in a 6% CO2 tissue culture incubator. Approximately 150,000 cells were loaded into triplicate Cell Culture Inserts. Migratory cells were subsequently dislodged and labeled as described in the Detailed Protocol. Migration overnight results in a two-fold increase in the number of migratory cells. Background migration (absence of serum) is low at both time points.

          Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
          InnoCyte™ and Interactive Pathways™ are trademarks of EMD Chemicals, Inc.