Abstract
The in vitro cell adhesion assay is a quantitative method for measuring selective cell adhesion to specific proteins. Traditionally, cell adhesion assays employ purified protein immobilized on a solid glass or plastic surface. Here, we describe a transient 293T cell transfection-based cell adhesion assay to study selective cell adhesion of a specific cell type to a protein of interest. In this protocol, 293T cells are transfected with a mammalian expression plasmid containing mSiglec1 cDNA or an empty plasmid as a mock control and are then cultured to form a monolayer. Subsequently, these Siglec1-expressing and mock-transfected 293T cell monolayers are used for cell adhesion assays with GFP-expressing B16F10 cells. The number of GFP+ cancer cells adhering to each 293T monolayer is a quantitative mean to compare the selective adhesiveness of cancer cells to Siglec1. This method eliminates the need to express and purify the protein of interest to perform in vitro cell adhesion assays and can easily be performed with difficult-to-purify proteins while maintaining their native in situ structure.
Highlights
Renders conventional cell adhesion assays inefficient in such situations
It is cumbersome to perform cell adhesion assays with difficult-to-purify proteins
We systematically describe the experimental procedures of 293T cell transient transfection-based cell adhesion assay adapted from our previously published work (Singh and Choi, 2019)
Summary
Renders conventional cell adhesion assays inefficient in such situations. it is cumbersome to perform cell adhesion assays with difficult-to-purify proteins. Enzyme-free PBS based cell dissociation buffer (Gibco, catalog number: 13151014) 12. 3. Incubate the cells at 37 °C in a 5% CO2 incubator for 2-5 min, add 3 ml of prewarmed complete DMEM, and transfer the cell suspensions to a 15 ml sterile conical tube.
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