Fluorescent Multiplex Cell Rolling Assay: Simultaneous Capturing up to Seven Samples in Real-Time Using Spectral Confocal Microscopy

Abstract

The parallel plate flow chamber assay is widely utilized to study physiological cell–cell adhesive interactions under dynamic flow that mimics the bloodstream. In this technique, the cells are perfused under defined shear stresses over a monolayer of endothelial cells (expressing homing molecules, e.g., selectins) or a surface (expressing recombinant homing molecules). However, with the need to study multiple samples and multiple parameters per sample, using a traditional bright-field microscope-based flow assay allows only one sample at a time to be analyzed, resulting in high interexperiment variability, the need for normalization, waste of materials, and significant consumption of time. We developed a multiplexing approach using a three-color fluorescence staining method, which allowed for up to seven different combination signatures to be run at one time. Using this fluorescent multiplex cell rolling (FMCR) assay, each sample is labeled with a different signature of emission wavelengths and mixed with other samples just minutes before the flow run. Subsequently, real-time images are acquired in a single pass using a line-scanning spectral confocal microscope. To illustrate the glycan-dependent binding of E-selectin, a central molecule in cell migration, to its glycosylated ligands expressed on myeloid-leukemic cells in flow, the FMCR assay was used to analyze E-selectin–ligand interactions following the addition (fucosyltransferase-treatment) or removal (deglycosylation) of key glycans on the flowing cells. The FMCR assay allowed us to analyze the cell-adhesion events from these different treatment conditions simultaneously in a competitive manner and to calculate differences in rolling frequency, velocity, and tethering capability of cells under study.

CD34+ HSPCs-derived exosomes contain dynamic cargo and promote their migration through functional binding with the homing receptor E-selectin

“…In particular, we uncovered several proteins involved in the migration of numerous cell types such as selectin ligands, chemokines and integrins ( Supplementary Table S3 ) suggesting that similar adhesion and migration functions could take place in exosomes as in their parental cells ( Figure 1C ). Here using as a model, the CD34 + AML cell line KG1a, a cell line previously described to possess optimum cell migration characteristics both in vitro and in vivo ( Dimitroff et al, 2001 ; Merzaban et al, 2011 ; AbuSamra et al, 2015 ; AbuSamra et al, 2017 ; Ali et al, 2017 ; AbuZineh et al, 2018 ; AbuElela et al, 2020 ; Aleisa et al, 2020 ; Al Alwan et al, 2021 ), we investigated the dynamic potential of KG1a-derived exosomes to interact with E-selectin. Our data were further confirmed using exosomes derived from healthy and AML primary CD34 + HSPCs cells.…”

Advances in Surface Enhanced Raman Spectroscopy for in Vivo Imaging in Oncology

“…The commonly used medical imaging modalities have limited capacity for multiplexed imaging: the radionuclides used in PET offer high sensitivity, but they provide photons of the same energy which are thus indistinguishable; CT contrast agents do not have sufficient sensitivity of detection to allow molecularly specific imaging; MRI relies primarily on intrinsic contrast, which again makes the detection of rare and microscopic targets practically impossible. In the preclinical setting, while a higher number of targets (i.e., up to five targets) can be detected using fluorescence imaging, [143][144][145] it is still mostly limited to in vitro setting and several issues, such as overlapping broad emission spectra, photobleaching, and autofluorescence, have complicated its in vivo implementation.…”

Nanoscopic Characterization of Cell Migration under Flow Using Optical and Electron Microscopy

“…To date, extensive studies have been conducted to understand the homing mechanisms of HSPCs, including initial rolling and tethering steps. ,, The most common and widespread technique to image and characterize rolling HSPCs has been a fluidics-based in vitro cell-rolling assay which mimics the cell-rolling behavior by flowing HSPCs in a fluidic chamber whose surface is coated with adhesion molecules. ,,− ,− Integration of advanced fluorescence imaging techniques to the microfluidics-based cell-rolling assay has enabled visualization and characterization of nanoscopic spatiotemporal dynamics of adhesion molecules as well as cell surface architecture during cell rolling. , Using this platform, we have demonstrated that the initial step of homing is regulated by spatial localization of the selectin ligands to membrane tethers and slings and their fast motion along these structures is due to the absence of anchoring to the underlying actin cytoskeleton. , Alternatively, the migration behavior of HSPCs has been investigated at the single-cell level by engineering endothelial vascular networks that closely mimic the bone marrow microenvironment . Cell rolling as well as cell extravasation of leukocytes − and HSPCs ,− across the endothelium have been studied using this approach.…”