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96-Well Plate
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48-602MAG
Buffer Detection Kit for Magnetic Beads
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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 Number
CBA010
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
References
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.
Product Information
Detection method
Fluorometric
Form
96 Tests
Format
96-well plate
Kit contains
Sterile 96-Well Migration Chamber, Culture Tray, Cell Detachment Buffer, Fluorescent Dye, Latrunculin A (inhibitor), and a user protocol.
The 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 Conditions
Upon 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 contains
Sterile 96-Well Migration Chamber, Culture Tray, Cell Detachment Buffer, Fluorescent Dye, Latrunculin A (inhibitor), and a user protocol.
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.
Upon arrival store the Calcein-AM at -20°C and the remaining components at 4°C.
Intended use
The 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.
Background
Cell 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 assay
The 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 protocol
1. 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 references
Loberg, R.D., et al. 2006. Neoplasia8, 578.
Registered Trademarks
Calbiochem® is a registered trademark of EMD Chemicals, Inc. InnoCyte™ and Interactive Pathways™ are trademarks of EMD Chemicals, Inc.
