A new adhesion assay using buoyancy to remove non-adherent cells

Cell adhesion assays are commonly used to study interactions between cells and substrates. Traditional methods rely on washing or centrifugation to separate adherent from non-adherent cells. However, these techniques may disrupt delicate adhesion events or introduce artifacts. Prior research has shown that washing can dislodge weakly adherent cells, potentially leading to underestimation of true binding. No prior work had resolved the issue of reliably removing non-adherent cells without affecting adhesion dynamics. This gap motivated the development of alternative methods. The buoyancy-based approach offers a novel solution by leveraging physical properties of cell suspensions. This study introduces a buoyancy-based adhesion assay as a potential improvement over conventional washing methods. The buoyancy method allows for the selective removal of non-adherent cells without mechanical disruption. This approach could enhance the accuracy of adhesion measurements in various biological contexts.

The purpose of this study was to develop and validate a new adhesion assay that uses buoyancy to remove non-adherent cells. The researchers aimed to compare this method with traditional washing techniques. They hypothesized that buoyancy could more effectively detect weak adhesion events. The study focused on cell-to-protein and cell-to-cell adhesion interactions. The buoyancy method was tested with biotinylated cells and various substrates. The goal was to determine if buoyancy could detect adhesion at lower substrate concentrations. The researchers also sought to assess the method's sensitivity in detecting cell-to-cell adhesion. This approach could improve the reliability of adhesion assays in biomedical research.

The buoyancy-based adhesion assay involved biotinylated cells incubated with substrates in well plates. Non-adherent cells were removed by floatation on a dense Percoll solution. Adherent cells were fixed using a Percoll/glutaraldehyde fixative. The fixed cells were quantified via streptavidin: horseradish peroxidase chemistry. The method was compared side-by-side with traditional washing assays. Cell-to-protein adhesion was tested using fibronectin and laminin substrates. Cell-to-cell adhesion was assessed with mononuclear leukocytes and KM12-L4 cells. The assay was also applied to human promyelocytic leukemia HL60 cells interacting with HUVEC.

The buoyancy method detected B16F10 binding to fibronectin at a 4-fold lower concentration than washing assays. It also detected HUVEC binding to laminin at a 10-fold lower concentration. In cell-to-cell adhesion, the buoyancy method showed significantly higher binding of mononuclear leukocytes and KM12-L4 cells to IL-1 beta treated HUVEC. HL60 binding to control and IL-1 beta treated HUVEC was approximately 60% with buoyancy. Washing assays showed an 8-fold higher binding (51% vs. 6%) for HL60 on IL-1 beta treated cells. The buoyancy method detected weak adhesion events that washing assays missed. This method proved more sensitive in low-background adhesion scenarios. These findings suggest buoyancy is a viable alternative to washing in adhesion assays.

The buoyancy-based adhesion assay demonstrated greater sensitivity in detecting weak adhesion events. It outperformed washing assays in detecting cell-to-protein adhesion at lower substrate concentrations. The method was also effective in detecting cell-to-cell adhesion when background binding was low. The buoyancy method did not alter adhesion dynamics during non-adherent cell removal. It reliably detected HL60 binding to HUVEC without overestimating interactions. The buoyancy method may be particularly useful for studying weak adhesion events. The authors suggest this approach could improve adhesion measurements in biomedical research. The buoyancy method offers a novel alternative to traditional washing techniques.