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The InnoCyte™ Monoctye Cell Migration Assay is designed to study the effects of various drugs or agents on monocyte motility or for identifying chemoattractant molecules. The assay is not intended for adherent cell lines, such as fibroblasts or solid tumor cell lines.
Catalogue Number
CBA098
Brand Family
Calbiochem®
Materials Required but Not Delivered
• Fluorescence plate reader capable of measuring fluorescence in 96-well plates at an excitation wavelength of ~485 nm and an emission wavelength of ~520 nm • Pipettes • PBS for washing the cells
References
References
Cravens, P.D. 2007. Scan. J. Immunol.65, 514. Charo, I.F., et al. 2006. N. Engl. J. Med.354, 610. Knitscher, U., et al. 2000. Eur. J. Pharmacol.401, 259. Turner, L., et al. 1999. Eur. J. Immunol.29, 2288. Cross, A.K., et al. 1997. Cytokine9, 521.
Product Information
Detection method
Fluorometric
Form
96 Tests
Format
96-Well Plate
Kit contains
96-Well Monocyte Cell Migration Chamber, Black, Conical 96-Well Plate, D-PBS, Calcein-AM, and a user protocol.
In case of contact with eyes, rinse immediately with plenty of water and seek medical advice. Wear suitable protective clothing.
Product Usage Statements
Intended use
The Calbiochem® InnoCyte™ 96-Well Monoctye Cell Migration Assay is intended for the study of the effects of various drugs or agents on monocyte motility or for identifying chemoattractant molecules. The assay is not intended for adherent cell lines, such as fibroblasts or solid tumor cell lines. To study these cell types, please use Cat. No. CBA010 or CBA017.
Storage and Shipping Information
Ship Code
Multiple Storage Conditions
Toxicity
Irritant
Storage
Multiple storage requirements
Storage Conditions
Upon arrival store the Microplates at room temperature, 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 contains
96-Well Monocyte Cell Migration Chamber, Black, Conical 96-Well Plate, D-PBS, Calcein-AM, and a user protocol.
Cravens, P.D. 2007. Scan. J. Immunol.65, 514. Charo, I.F., et al. 2006. N. Engl. J. Med.354, 610. Knitscher, U., et al. 2000. Eur. J. Pharmacol.401, 259. Turner, L., et al. 1999. Eur. J. Immunol.29, 2288. Cross, A.K., et al. 1997. Cytokine9, 521.
用户协议
Revision
11-December-2022 JSW
Form
96 Tests
Format
96-Well Plate
Detection method
Fluorometric
Storage
Upon arrival store the Microplates at room temperature, the Calcein-AM at -20°C and the remaining components at 4°C.
Intended use
The Calbiochem® InnoCyte™ 96-Well Monoctye Cell Migration Assay is intended for the study of the effects of various drugs or agents on monocyte motility or for identifying chemoattractant molecules. The assay is not intended for adherent cell lines, such as fibroblasts or solid tumor cell lines. To study these cell types, please use Cat. No. CBA010 or CBA017.
Background
Cell migration is critical for embryonic development, the inflammatory immune response, wound repair, and tumor formation. Circulating monocytes are precursors for phagocytes, such as macrophages and dendritic cells. In immune responses, monocyte migration is critical for providing immunological defense and eliminating infectious agents. Monocytes circulate in the blood until encountering specific chemotactic factors that initiate the migration process.
Inflammatory disorders has become a focus area for development of pharmaceutical agents specific for a wide range of novel targets. Among these new targets are chemokines, proteins that attract cells of the immune system to sites of inflammation. The largest chemokine family consists of CC chemokines, so named because the first two of the four cysteine residues in these molecules are adjacent to each other. CC chemokines attract mononuclear cells to sites of chronic inflammation, such as sites of trauma, bacterial infection, toxin exposure, and ischemia. The monocyte chemoattractant proteins consist of a group of related peptides - CCL2, CCL7, CCL8, and CCL13 - encoded on chromsome 17. The CCR2 chemokine receptor is the only known receptor for CCL2 (monocyte chemoattactant protein-1) and CCL13. CCR2 and its primary ligand, monocyte chemoattractant protein-1 (MCP-1), are the most popular targets for drug development. These two factors play a key role in attracting monocytes to inflammatory sites, where the monocytes differentiate to become macrophages that secrete inflammatory cytokines like TNF-α. Clinical examples include multiple sclerosis (MS) and atherosclerosis. In the lesions of MS, MCP-1 is expressed in astrocytes, while infiltrating monocytes/macrophages express CCR2. In atherosclerosis, CCL2 is present in macrophage-rich atherosclerotic plaques. Several CCR2 antagonists are currently in clinical trials.
Principles of the assay
The Calbiochem® InnoCyte™ 96-Well Monocyte Cell Migration Assay is provided in a convenient 96-well format. The assay is based on the Boyden-chamber principle. Each well of the 96-well cell culture insert contains a polycarbonate membrane with a 5 µm pore size, which is suitable for monocytes. Cells are placed in the wells of the upper chamber in serum-free medium, while medium containing serum or chemotactic substances is placed in the wells of the lower chamber. Migrated cells from the wells of the lower chamber are transferred to wells of a black 96-well plate and labeled with calcein-AM. Fluorescence is determined using a 96-well plate fluorimeter with an excitation wavelength of ~485 nm and an emission wavelength of ~520 nm. The fluorescence intensity is proportional to the number of migrated cells.
• Fluorescence plate reader capable of measuring fluorescence in 96-well plates at an excitation wavelength of ~485 nm and an emission wavelength of ~520 nm • Pipettes • PBS for washing the cells
Reagent preparation
Cell Labeling Mixture: Warm the D-PBS and Calcein-AM Solution to room temperature. Add 35 µl Calcein-AM Solution to 10 ml D-PBS. Mix well. Note: Prepare just before use (see step 7 of the Detailed Protocol below).
Detailed protocol
1. Harvest cells and wash 2 times with PBS. 2. Resuspend the cell pellet in serum-free medium or D-PBS to a final cell concentration of ~ 2-4 x 106 cells/ml. 3. Remove the cell culture insert (upper chamber) from the 96-well tray (lower chamber) of the 96-Well Monocyte Cell Migration Chamber. 4. Add 150 µl appropriate serum-free cell culture medium containing desired chemoattractants (e.g., serum or chemokines) to the lower chamber. Prepare a negative control with cell culture medium without serum or other chemoattractants. 5. Place the cell culture inserts (upper chamber) in the 96-well tray (lower chamber) and add 100 µl of the cell suspension to appropriate wells of the cell culture insert (upper chamber); replace the lid. 6. Incubate for 2-20 h in a CO2 cell culture incubator. 7. At the end of the incubation period in step 6, prepare the Cell Labeling Mixture as outlined under Reagent Preparation. 8. Following the incubation in step 6, remove the upper chamber and discard it. 9. Transfer the medium containing cells from the wells of the lower chamber to wells in the Black, Conical 96-Well Plate with a multi-channel pipette. 10. Place the Black, Conical 96-Well Plate containing the cells in a bench-top centrifuge equipped with plate holders and centrifuge for 5-10 min at 250-300 x g at 10-25°C. 11. Discard the supernatant by shaking the inverted plate over the sink once or twice without dislodging the pelleted cells at the bottom of the wells. 12. Add 200 µl PBS to each well and then discard gently (small volume left in the well is fine); do not resuspend the cells. Tap the inverted plate gently on a stack of paper towels. 13. Add 100 µl Cell Labeling Mixture to each well, pipette up and down several times to obtain a homogenous cell suspension, and cover the plate with a Plate Sealer. 14. Incubate for 30-60 min at 37°C in the CO2 incubator. 15. Read the fluorescence using 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: Serum Mediated Migration of THP-1 and U937 Cells
40,000 THP-1 or U937 cells were incubated in triplicate wells of the upper chamber and allowed to migrate toward medium with or without 10% serum in the lower chamber for 3 h in a 6% CO2 incubator at 37°C. The cells were labeled as outlined in the Detailed Protocol.
Figure 2: MCP-1 and Serum Mediated Migration of THP-1 Cells
40,000 THP-1 cells were incubated in triplicate wells of the upper chamber and allowed to migrate toward medium with or without chemotactic agent in the lower chamber for 3 h in a 6% CO2 incubator at 37°C. The cells were labeled as outlined in the Detailed Protocol.
Registered Trademarks
Calbiochem® is a registered trademark of EMD Millipore, Inc. InnoCyte™ and Interactive Pathways™ are trademarks of EMD Millipore, Inc.
