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ECM558 Sigma-AldrichQCM™ Tumor Cell Transendothelial Migration Assay (Colorimetric, 24 Assays)

This QCM Tumor Cell Transendothelial Cell Migration Assay -Colorimetric provides an efficient model to analyze the ability of tumor cells to invade the endothelium.

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ECM558

1 kit 24 Assays

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Key Spec Table

Detection Methods
Colorimetric

Description
Catalogue Number	ECM558
Trade Name	QCM Chemicon
Description	QCM™ Tumor Cell Transendothelial Migration Assay (Colorimetric, 24 Assays)
Overview	Also available: Cell Comb™ Scratch Assay! Get biochemical data from a scratch assay! Click Here Introduction A quantitative assay for tumor transendothelial migration has been described using a modified Boyden chamber system, (Okada, 1994; Li, 1999; Laferriere, 2001). 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. A tumor cell suspension is added above the endothelial monolayer. The invasion of tumor cells across the endothelium is determined by measuring the number of cells that migrate to the lower chamber. Millipore's QCM Tumor Cell Transendothelial Cell Migration Assay - Colorimetric provides an efficient model to analyze the ability of tumor cells to invade the endothelium. The assay is designed with an 8 µm pore size cell culture insert, appropriate for most cancer cell lines. 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 compare the invasiveness of a variety of tumor cell lines, and to evaluate the effects of various factors influencing the process. Precoated cell culture inserts are provided in the Millipore QCM Tumor Cell Transendothelial Cell Migration Assay to significantly reduce assay time. Additionally, the assay allows quantitative analysis of tumor cell migration. Following incubation of tumor cells with the endothelial cell layer, invasive tumor cells are stained and quantified. In a departure from traditional Boyden methodology, stain is eluted with extraction buffer, transferred to a microplate, and measured spectrophotometrically. (Prior to elution, the investigator has the option of counting cells individually, if desired.) Spectrophotometric absorbance correlates with cell migration.
Materials Required but Not Delivered	1. Endothelial cells (for example: HUVECs), and cell culture medium (for example: EGM-2) 2. Invasive tumor cell lines and cell culture medium 3. 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 4. Serum-free medium, such as DMEM, MEM, etc. containing 0.5% BSA 5. Sterile PBS or HBSS to wash cells 6. Distilled water 7. (Optional) Chemoattractant or pharmacological agent for addition to culture medium 8. Low speed centrifuge and tubes for cell harvesting 9. CO2 incubator appropriate for subject cells 10. Hemocytometer or other means of counting cells 11. Trypan blue or equivalent viability stain 12. Microplate reader (540-570 nm detection) or spectrophotometer 13. (Optional) Graduated ocular (calibrated), or automated method for counting stained cells on a membrane
Background Information	Tumor metastasis is a multistep cascade. In order for tumor cells to migrate from a primary tumor mass to distant locations, they must invade through the basal membrane and into blood vessels (intravasation), circulate in the blood stream, survive during transport, then migrate out of a blood vessel (extravasation) to establish micrometastases. The penetration of circulating tumor cells into the endothelium is a crucial step for tumor metastasis.

References

Product Information
Components	1. Migration Test Plate: (Part No. CS202627) Two 24-well culture plates, each containing 12 human fibronectin-coated cell culture inserts (8 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. Extraction Buffer: (Part No. 20295) One vial - 10 mL 5. 24 well Stain Extraction Plate: (Part No. 2005871) One plate. 6. 96 well Stain Quantitation Plate: (Part No. 2005870) One plate. 7. Swabs: (Part No. 10202) 50 each. 8. Forceps: (Part No. 10203) 1 pair.
Detection method	Colorimetric
Quality Level	MQ100

Applications
Application	This QCM Tumor Cell Transendothelial Cell Migration Assay -Colorimetric provides an efficient model to analyze the ability of tumor cells to invade the endothelium.
Application Notes	Calculation of Results Results of the assay may be illustrated graphically using a bar chart to display optical density (OD) values at ~570 nm. A typical tumor cell transendothelial migration experiment will compare with a negative control. Negative control chambers, containing HUVECs only, function to determine the level of HUVECs migrating through the membrane. Cell migration in these chambers is generally low as there is no ECM on the lower side of the membrane to support endothelial migration, and these chambers are typically used as "blanks" for interpretation of data. As such, migration in test wells can be described as the value of tumor transendothelial migration minus the amount of migration visualized in the HUVEC only control. Additional migration may also be induced or inhibited in test wells through the addition of cytokines or other pharmacological agents. The following figures demonstrate typical migration results. One should use the data at left for reference only. This data should not be used to interpret actual assay results.

Applications

Application

This QCM Tumor Cell Transendothelial Cell Migration Assay -Colorimetric provides an efficient model to analyze the ability of tumor cells to invade the endothelium.

Application Notes

Calculation of Results

Results of the assay may be illustrated graphically using a bar chart to display optical density (OD) values at ~570 nm.

A typical tumor cell transendothelial migration experiment will compare with a negative control. Negative control chambers, containing HUVECs only, function to determine the level of HUVECs migrating through the membrane. Cell migration in these chambers is generally low as there is no ECM on the lower side of the membrane to support endothelial migration, and these chambers are typically used as "blanks" for interpretation of data. As such, migration in test wells can be described as the value of tumor transendothelial migration minus the amount of migration visualized in the HUVEC only control.

Additional migration may also be induced or inhibited in test wells through the addition of cytokines or other pharmacological agents.
The following figures demonstrate typical migration results. One should use the data at left for reference only. This data should not be used to interpret actual assay results.

Biological Information

Physicochemical Information

Dimensions

Materials Information

Toxicological Information

Safety Information according to GHS

Safety Information

Product Usage Statements
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.

Storage and Shipping Information
Storage Conditions	TNFα should be stored at -20°C. All other components should be store at 2° to 8°C. Please refer to kit label for expiration date.

Packaging Information
Material Size	1 kit
Material Package	24 Assays

Transport Information

Supplemental Information

Specifications

Global Trade Item Number
Catalogue Number	GTIN
ECM558	04053252014758

Documentation

QCM™ Tumor Cell Transendothelial Migration Assay (Colorimetric, 24 Assays) SDS

Title
Safety Data Sheet (SDS)

References

Reference overview	Pub Med ID
Transendothelial migration of colon carcinoma cells requires expression of E-selectin by endothelial cells and activation of stress-activated protein kinase-2 (SAPK2/p38) in the tumor cells. Laferriere, J, et al. J. Biol. Chem., 276: 33762-72 (2001) 2001 Show Abstract Adhesion and migration of tumor cells on and through the vascular endothelium are critical steps of the metastatic invasion. We investigated the roles of E-selectin and of stress-activated protein kinase-2 (SAPK2/p38) in modulating endothelial adhesion and transendothelial migration of HT-29 colon carcinoma cells. Tumor necrosis factor alpha (TNF alpha) strongly increased the expression of E-selectin in human umbilical vein endothelial cells (HUVEC). This effect was independent of the activation of SAPK2/p38 induced by TNF alpha. Adhesion of HT-29 cells on a monolayer of HUVEC pretreated with TNF alpha was dependent on E-selectin expression but was independent of SAPK2/p38 activity of both HUVEC and tumor cells. The adhesion of HT-29 cells to E-selectin-expressing HUVEC led to the activation of SAPK2/p38 in the tumor cells as reflected by the increased phosphorylation of the actin-polymerizing factor HSP27 by mitogen-activated protein kinase 2/3, a direct target of SAPK2/p38. Moreover, a recombinant E-selectin/Fc chimera quickly increased the activation of SAPK2/p38 in HT-29 cells. Blocking the increased activity of SAPK2/p38 of HT-29 cells by SB203580 or by expressing a dominant negative form of SAPK2/p38 inhibited their transendothelial migration. Similarly, HeLa cells stably expressing a kinase-inactive mutant of SAPK2/p38 showed a decreased capacity to cross a layer of HUVEC. Overall, our results suggest that the regulation of transendothelial migration of tumor cells involves two essential steps as follows: adhesion to the endothelium through adhesion molecules, such as E-selectin, and increased motogenic potential through adhesion-mediated activation of the SAPK2/p38 pathway.	11448946
A modified Boyden chamber assay for tumor cell transendothelial migration in vitro. Li, Y H and Zhu, C Clin. Exp. Metastasis, 17: 423-9 (1999) 1999	10651309
A novel in vitro assay system for transendothelial tumor cell invasion: significance of E-selectin and alpha 3 integrin in the transendothelial invasion by HT1080 fibrosarcoma cells. Okada, T, et al. Clin. Exp. Metastasis, 12: 305-14 (1994) 1994	7518760

Brochure

Title
Advancing cancer research: From hallmarks & biomarkers to tumor microenvironment progression
Cell Migration and Invasion: Choosing the Right Assay

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ECM558 Sigma-AldrichQCM™ Tumor Cell Transendothelial Migration Assay (Colorimetric, 24 Assays)

This QCM Tumor Cell Transendothelial Cell Migration Assay -Colorimetric provides an efficient model to analyze the ability of tumor cells to invade the endothelium.

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