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CBA010 InnoCyte™ Cell Migration Assay, 96-well

InnoCyte™ Cell Migration Assay, 96-well MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.
CBA010
  
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      Overview

      Replacement Information

      Key Spec Table

      Detection Methods
      Fluorometric
      Description
      Overview

      This product has been discontinued.



      A convenient assay for the chemotaxis cell migration. 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 (Excitation max.: ~485 nm; Emission max.: ~520 nm).

      Catalogue NumberCBA010
      Brand Family Calbiochem®
      Application Data
      ~50,000 human umbilical venous endothelial cells (HUVECs) were incubated in triplicate wells of the upper chamber and allowed to migrate toward medium with or without chemotactic components in the lower chamber for 4 h in a 6% CO2 incubator at 37°C. The cells were detached and labeled as indicated in the Detailed Protocol. Latrunculin A, a powerful inhibitor of actin polymerization, was added to an aliquot of cell suspension immediately prior to loading the cells in the wells.

      ~60,000 HT-1080 cells were incubated in triplicate wells of the upper chamber and allowed to migrate toward medium with or without serum for 3 h in a 6% CO2 incubator at 37°C. The cells were detached and labeled as indicated in the Detailed Protocol.
      Materials Required but Not Delivered• Pipettors with disposable tips
      • Black 96-well strips or black plate
      • Phosphate buffered saline (PBS)
      • Fluorescence reader capable of measuring fluorescence in 96-well plates at an excitation wavelength of ~485 nm and an emission wavelength of ~520 nm
      References
      ReferencesKlinghoffer, 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.
      Lauffenburger, D.A., and Horowitz, A.F. 1996. Cell 84, 359.
      Product Information
      Detection methodFluorometric
      Form96 Tests
      Format96-well plate
      Kit containsSterile 96-Well Migration Chamber, Culture Tray, Cell Detachment Buffer, Fluorescent Dye, Latrunculin A (inhibitor), and a user protocol.
      Quality LevelMQ100
      Applications
      Application ReferencesLoberg, R.D., et al. 2006. Neoplasia 8, 578.
      Biological Information
      Assay time3-5 h
      Sample TypeAdherent cells
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Intended useThe Calbiochem® InnoCyte™ Cell Migration Assay is designed to study the effects of various drugs or agents on cell motility or for identifying chemoattractant molecules (chemotactic migration). The assay is not intended for leukocyte migration experiments, as the study of leukocytes requires membranes with a pore-size smaller than 8 µm.
      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 containsSterile 96-Well Migration Chamber, Culture Tray, Cell Detachment Buffer, Fluorescent Dye, Latrunculin A (inhibitor), and a user protocol.
      Specifications
      Global Trade Item Number
      Catalogue Number GTIN
      CBA010 0

      Documentation

      InnoCyte™ Cell Migration Assay, 96-well MSDS

      Title

      Safety Data Sheet (SDS) 

      InnoCyte™ Cell Migration Assay, 96-well Certificates of Analysis

      TitleLot Number
      CBA010

      References

      Reference overview
      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.
      Lauffenburger, D.A., and Horowitz, A.F. 1996. Cell 84, 359.

      Citations

      Title
      Nawshad, A., et al. 2007. Journal of Cell Science 120, 1646. Deng, D.XF., et al. 2006. Arteriosclerosis, Thrombosis, and Vascular Biology 26 1058. Fosbrink M., et al. 2006. J. Biol. Chem. 281, 19009. Loberg, R.D., et al. 2006. Neoplasia 8, 578.
      User Protocol

      Revision26-July-2011 RFH
      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 Calbiochem® InnoCyte™ Cell Migration Assay is designed to study the effects of various drugs or agents on cell motility or for identifying chemoattractant molecules (chemotactic migration). The assay is not intended for leukocyte migration experiments, as the study of leukocytes requires membranes with a pore-size smaller than 8 µm.
      BackgroundCell migration is crucial for embryonic development, the inflammatory immune response, wound repair, and tumor formation and metastasis. Recent findings highlight the importance of tension, the actin and myosin filament network, the Rho/Rac family of signaling molecules, and microtubules in cell migration. The coordination of these complex processes is the challenge facing cells that are on the move. The regulation of focal adhesion and focal complex turnover is critical for the continued remodeling and reorganization of adhesion contacts during cell migration, and migratory defects have been reported in cells lacking Src family kinases, focal adhesion kinase (FAK), and calpain. When the cells in a tumor develop the ability to migrate out of the initial tumor and invade their surroundings, they become very dangerous. Some cells may migrate into the circulatory system and move to new locations (metastasis), where they then form a new (secondary) tumor. This change in the cells comprising a tumor, from sedentary to migratory, represents a serious problem, one that may be solved with a better understanding of the migratory process itself.
      Principles of the assayThe Innocyte™ Cell Migration Assay is provided in a convenient 96-well format suitable with cell culture inserts containing a membrane with an 8 µm pore-size that ensures non-specific, random migration is minimized. Cells are placed in the cell culture inserts in serum-free medium, while medium containing serum or other factors is placed in the wells of the plate; together the inserts and the plate form the Cell Migration Chamber. Cells that migrate through the membrane attach to the underneath side of the cell culture insert and are subsequently detached using Cell Detachment Buffer containing Calcein-AM fluorescent dye. The results are read using a standard fluorescent plate reader and results quantitated.

      Note: The membrane is not coated with extracellular matrix protein(s), and is therefore not suitable for the study of haptotactic migration. There is no need for manual cell scraping or cell counting.
      Materials provided• 96-well Cell Migration Chamber (Kit Component No. JA7830): 1 sterile 96-well plate with cell culture inserts and tray
      • Additional 96-Well Tray (Kit Component No. JB306): 1 plastic tray for cell detachment
      • Cell Detachment Buffer (Kit Component No. JA7709): 1 bottle, 20 ml
      • Calcein-AM Solution (Kit Component No. JA7705): 2 amber vials, 50 µl each
      • Latrunculin A Solution (Antimigratory Compound) (Kit Component No. JA7832): 1 amber vial, 25 µl, supplied at 1 mM
      Materials Required but not provided• Pipettors with disposable tips
      • Black 96-well strips or black plate
      • Phosphate buffered saline (PBS)
      • Fluorescence reader capable of measuring fluorescence in 96-well plates at an excitation wavelength of ~485 nm and an emission wavelength of ~520 nm
      Reagent preparation• Labeling/Cell Detachment Mixture: a. Warm Cell Detachment Buffer and Calcein-AM Solution to room temperature. b. Add 65 µl Calcein-AM Solution to 20 ml of cell detachment buffer. Mix well.
      Detailed protocol1. Harvest cells and wash 2 times with PBS.
      2. Resuspend the cell pellet with appropriate serum-free cell culture medium to a final cell concentration of approximately 250,000-500,000 cells/ml.
      3. Remove the 96-well cell culture insert (upper chamber) from the 96-well tray (lower chamber).
      4. Add 150-200 µl appropriate serum-free cell culture medium containing desired chemoattractant(s) (e.g. serum or growth factors) to the lower chamber. Prepare a negative control with cell culture medium without serum or other chemoattractant(s).
      5. Return the upper chamber to the lower chamber and add 100-150 µl cell suspension prepared in step 2 to designated wells. If desired compounds that affect cell motility may be added to an aliquot of cell suspension at the desired concentration immediately prior to loading the cells in the wells. If desired, add the Latrunculin A Solution (provided) to a final concentration of 1-3 µM as a migratory inhibitor control.
      6. Replace the lid and incubate the migration chamber for 4-24 h in a CO2 incubator at 37°C.
      7. Add 200 µl Labeling/Cell Detachment solution to corresponding wells of the Additional 96-well Tray.
      8. Remove the migration chamber from the CO2 incubator and gently remove the upper chamber and flick the medium and cells from the wells.
      Note: It will not affect the assay if residual medium or cells remain in the wells of the upper chamber.
      9. Place the upper chamber in the Additional 96-well Tray containing the Labeling/Detachment Mixture.
      Note: The cells that have migrated through the membrane in the upper chamber will cling to the underneath side of the membrane so the bottom surface of the membrane should not be touched or placed on the workspace during the transfer.
      10. Replace the lid and incubate the migration chamber for 20-30 min in a CO2 incubator at 37°C.
      11. Remove the migration chamber from the incubator and gently tilt the upper chamber, while its bottom side still engaged in the solution of the lower chamber, back and forth several times to facilitate the dislodgement process.
      12. Remove the upper chamber and discard. Place the lid over the Additional 96-well Tray and incubate for 30-45 min in the CO2 incubator at 37°C.
      13. Remove the Additional 96-well Tray containing the labeled cells from the CO2 incubator and transfer 150 µl from each well corresponding wells of a black 96-well plate. Measure the fluorescence in a fluorescence plate reader set at an excitation wavelength of ~485 nm and an emission wavelength of ~520 nm.
      Assay characteristics and examples

      Figure 1: Assay Characteristics and Examples

      ~50,000 human umbilical venous endothelial cells (HUVECs) were incubated in triplicate wells of the upper chamber and allowed to migrate toward medium with or without chemotactic components in the lower chamber for 4 h in a 6% CO2 incubator at 37°C. The cells were detached and labeled as indicated in the Detailed Protocol. Latrunculin A, a powerful inhibitor of actin polymerization, was added to an aliquot of cell suspension immediately prior to loading the cells in the wells.


      Figure 2: Assay Characteristics and Examples

      ~60,000 HT-1080 cells were incubated in triplicate wells of the upper chamber and allowed to migrate toward medium with or without serum for 3 h in a 6% CO2 incubator at 37°C. The cells were detached and labeled as indicated in the Detailed Protocol.
      Application referencesLoberg, R.D., et al. 2006. Neoplasia 8, 578.
      Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
      InnoCyte™ and Interactive Pathways™ are trademarks of EMD Chemicals, Inc.