Abstract
Parallel plate cell rolling flow assay is one of the widely used techniques to assess cell-cell adhesion interactions under dynamic conditions mimicking the physiologic situation. However, with the need to study multiple samples and multiple ligands, using the light microscope-based flow assay allows only one sample at a time which brings about high inter-experiment variability, need for normalization, waste of materials and is significantly time consuming. In order to develop a multiplexing technique we made use of a three-color fluorescence staining approach, which allows eight different combination signatures to be run at one time. Using this technique, each sample is labeled with a different signature of emission wavelengths and mixed with other samples before the flow run. Real-time images are acquired in a single pass using line-scanning spectral confocal microscope at one frame per 400-millisecond rate. To show the proof of principle of our multiplex assay approach, in this study, we confirmed the glycan-dependent binding of E-selectin to its ligands through various deglycosylation treatments [sialidase, O-sialoglycan endopeptidase (OSGE) and Peptide-NGlycosidase F (PNGase F)] of KG1a leukemic cells. Each cell treatment condition was labeled with a unique fluorescent signature comprised of Alexa Fluor 405-, Alexa Fluor 546- or Alexa Fluor 680-conjugated secondary antibody to anti-CD34 antibody or combinations thereof. Subsequently, E-selectin-dependent cell rolling activity on live E-selectin-expressing CHO (CHO-E) cells or mock-transfected CHO (CHO-M) cells (as negative control) was observed in real-time. This technique allowed us to analyze adhesion events from these eight different treatment conditions simultaneously in real-time and to calculate differences in rolling frequency, velocity and tethering capability of cells under study.