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Endothelial Cell Layer. Image depicts a confluent endothelial cell layer after 72 hour incubation on a cell culture insert. Cells are stained with Ce...
Transendothelial migration of HL-60 cells. HUVECs (1x105 cells) were grown to confluency for 72 hrs on cell culture inserts. HL-60 cells (2x105 cells)...
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QCM™ Leukocyte Transendothelial
Migration Assay (Colorimetric, 24 Assays)
Description:
QCM™ Leukocyte Transendothelial Migration Assay (Colorimetric, 24 Assays)
Trade Name:
Chemicon (Millipore)
Qty/Pk:
1 kit
Product Overview:
Millipore’s Colorimetric QCM™ Leukocyte Transendothelial Cell Migration Assay provides an efficient model to analyze the ability of leukocytes to migrate through the endothelium. The assay is designed with a 3 µm pore size cell culture insert, appropriate for most leukocyte migration. The upper side of the cell culture insert is coated with fibronectin to support the optimal attachment and growth of endothelial cells. The assay allows investigators to evaluate the effects of various factors that influence the transendothelial process.
Precoated cell culture inserts are provided in the Millipore QCM Leukocyte Transendothelial Cell Migration Assay to significantly reduce assay time. Additionally, the assay allows quantitative analysis of leukocyte migration. Following incubation of leukocytes with the endothelial cell layer, migratory cells are stained and quantified with WST-1 reagent and measured using a spectrophotometer. Absorbance correlates with cell migration.
Precoated cell culture inserts are provided in the Millipore QCM Leukocyte Transendothelial Cell Migration Assay to significantly reduce assay time. Additionally, the assay allows quantitative analysis of leukocyte migration. Following incubation of leukocytes with the endothelial cell layer, migratory cells are stained and quantified with WST-1 reagent and measured using a spectrophotometer. Absorbance correlates with cell migration.
Background Information:
Leukocytes patrol the vascular system. They must migrate across endothelial barriers in order to recruit to sites of inflammation. This process involves a multistep cascade consisting of leukocyte rolling, adhesion, and transmigration. A quantitative assay for leukocyte transendothelial migration has been described using a modified Boyden chamber system (Roth et al. 1995, Ding et al. 2000). The Boyden chamber system is a two chamber system with a porous membrane providing an interface between these two chambers. Endothelial cells are cultured on top of the porous membrane that is coated with an extracelluar matrix (ECM) protein. Once a confluent layer of cells is established, leukocytes are then added onto the endothelial monolayer. Leukocyte migration across the endothelium is determined by measuring the number of cells that migrate between the endothelial cells, through the porous membrane, to the lower chamber.
Application Notes:
Results of the assay may be illustrated graphically using a bar chart to display optical density (OD) values at 420 - 480 nm.
Additional migration may also be induced or inhibited in test wells through the addition of cytokines or other pharmacological agents.
Figures 2 and 3 demonstrate typical migration results. One should use the data for reference only. This data should not be used to interpret actual assay results.
Additional migration may also be induced or inhibited in test wells through the addition of cytokines or other pharmacological agents.
Figures 2 and 3 demonstrate typical migration results. One should use the data for reference only. This data should not be used to interpret actual assay results.
Usage Statement:
Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
Components:
- 1. Migration Test Plate: (Part No. CS202626) Two 24-well culture plates, each containing 12 human fibronectin-coated cell culture inserts (3 μm pore size), sufficient for the evaluation of 24 test samples
- 2. TNFα: (Part No. 2004167) One tube - 20 μL at 0.1 mg/mL
- 3. Cell Stain Solution: (Part No. 20294) One vial - 10 mL
- 4. WST-1 Reagent: (Part No. CS201370) One vial.
- 5. Electro Coupling Solution: (Part No. CS201382) One vial - 5 mL.
- 6. 96 well Stain Quantitation Plate: (Part No. 2005870) One plate.
- 7. Forceps: (Part No. 10203) 1 pair
Storage Conditions:
The kit is packaged in two individual boxes. ECM557-1 contains components that upon arrival should be stored at 2° to 8°C. ECM557-2 contains TNFα , WST-1, and Electro Coupling Solution that upon arrival should be stored at -20°C. Please refer to kit label for expiration date.
Detection Methods:
Colorimetric
Packaging:
24 Assays
Protein or Enzyme Type:
Extracellular Matrix Proteins
Product Name:
QCM™ Leukocyte Transendothelial
Migration Assay (Colorimetric, 24 Assays)
Migration Assay (Colorimetric, 24 Assays)
Materials Required but Not Delivered:
1. Endothelial cells (for example: HUVECs, Cat No. SCCE001)
2. Endothelial cell culture medium (Cat. No. SCME001)
3. Leukocytes and cell culture medium
4. Harvesting buffer: Millipore’s cell detachment solution, Accutase™ (Cat. No. SCR005), EDTA or trypsin-based cell detachment buffer, or other cell detachment formulations as optimized by individual investigators can also be used.
5. Serum-free medium, such as RPMI-1640, DMEM (Cat No. ES-101-B), MEM, etc. containing 0.5% BSA (Cat No. 82-046-4), Pen/Strep (Cat No. TMS-AB2-C).
6. Sterile PBS (Cat No. BSS-1006-B) to wash cells.
7. Distilled water (Cat No. TMS-006-B)
8. (Optional) Chemoattractant or pharmacological agent for addition to culture medium.
9. Low speed centrifuge and tubes for cell harvesting.
10. CO2 incubator appropriate for subject cells.
11. Microplate reader (420 - 480 nm detection) or a spectrophotometer.
2. Endothelial cell culture medium (Cat. No. SCME001)
3. Leukocytes and cell culture medium
4. Harvesting buffer: Millipore’s cell detachment solution, Accutase™ (Cat. No. SCR005), EDTA or trypsin-based cell detachment buffer, or other cell detachment formulations as optimized by individual investigators can also be used.
5. Serum-free medium, such as RPMI-1640, DMEM (Cat No. ES-101-B), MEM, etc. containing 0.5% BSA (Cat No. 82-046-4), Pen/Strep (Cat No. TMS-AB2-C).
6. Sterile PBS (Cat No. BSS-1006-B) to wash cells.
7. Distilled water (Cat No. TMS-006-B)
8. (Optional) Chemoattractant or pharmacological agent for addition to culture medium.
9. Low speed centrifuge and tubes for cell harvesting.
10. CO2 incubator appropriate for subject cells.
11. Microplate reader (420 - 480 nm detection) or a spectrophotometer.