The CytoSelect Tumor-Endothelium Adhesion Assay Kit (Cell Biolabs) was used to evaluate the adhesion potential of tumor cells on a brain endothelial cell monolayer. Briefly, 1 × 106 brain endothelial cells (hCMEC/D3) were seeded onto fibronectin (10 µg/mL) and grown to confluency. The endothelial cell monolayer was first treated with 25 pg/mL of TNF-α overnight to activate CD62E expression. Primary and secondary lung cancer cell lines were tagged by a green fluorescent dye (Cyto Tracker, Cell Biolabs). 1 × 105 cancer cells were then seeded onto the activated hCMEC/D3 monolayer for 90 minutes. Nonadherent cells were washed with PBS, and relative cell attachment was determined using a POLARstar OPTIMA microplate reader (BMG LABTECH,). The experiment was repeated 3 times in triplicate. In a separate method to evaluate adhesion, we used ICC and confocal image analysis (see above). The same conditions that were used in the CytoSelect adhesion assay were repeated, except on coverslips, and prepared for ICC. Semiquantification of adhesion was assessed using confocal images and Zeiss ZEN software.
Cytoselect tumor endothelium adhesion assay kit
Manufactured by Cell Biolabs
Sourced in United States
The CytoSelect Tumor-Endothelium Adhesion Assay Kit allows for the quantitative measurement of the adhesion of tumor cells to endothelial cells. It provides a standardized platform to study the interactions between tumor cells and the endothelium.
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2 protocols using cytoselect tumor endothelium adhesion assay kit
1
Tumor-Endothelial Adhesion Assay for Lung Cancer
2
Tumor Cell-Endothelial Adhesion Assay
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Adhesion ability of tumour cell–brain endothelial cell was evaluated using the CytoSelect™ Tumor-Endothelium Adhesion Assay Kit (Cell Biolabs, San Diego, CA, USA). Briefly, 48-well plate (Corning, Corning, NY, USA) was coated with 10 µg/mL of fibronectin (Sigma-Aldrich, Gillingham, UK). Then, 1 × 106 cells/well hCMEC/D3 were seeded and grown to 80% confluency. To activate the extracellular expression of CD62E, hCMEC/D3 cells were incubated at 25 pg/mL of TNF-α. In parallel, NSCLC cells were stained with live cell green fluorescent dye (Cyto Tracker™) (Cell Biolabs, San Diego, CA, USA) for 1 h. Then, 1 × 105 of the stained NSCLC cells were seeded onto the activated hCMEC/D3 monolayer and the co-culture incubated for 90 min. Non-adherent cells were washed with pre-warmed PBS and the relative number of adherent cancer cells was evaluated using a POLARstar™ OPTIMA microplate reader (BMG Labtech, Aylesbury, UK). All experiments were conducted independently three times in triplicate.
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