qFlow Cytometry-Based Receptoromic Screening: A High-Throughput Quantification Approach Informing Biomarker Selection and Nanosensor Development

Abstract

Nanosensor-based detection of biomarkers can improve medical diagnosis; however, a critical factor in nanosensor development is deciding which biomarker to target, as most diseases present several biomarkers. Biomarker-targeting decisions can be informed via an understanding of biomarker expression. Currently, immunohistochemistry (IHC) is the accepted standard for profiling biomarker expression. While IHC provides a relative mapping of biomarker expression, it does not provide cell-by-cell readouts of biomarker expression or absolute biomarker quantification. Flow cytometry overcomes both these IHC challenges by offering biomarker expression on a cell-by-cell basis, and when combined with calibration standards, providing quantitation of biomarker concentrations: this is known as qFlow cytometry. Here, we outline the key components for applying qFlow cytometry to detect biomarkers within the angiogenic vascular endothelial growth factor receptor family. The key aspects of the qFlow cytometry methodology include: antibody specificity testing, immunofluorescent cell labeling, saturation analysis, fluorescent microsphere calibration, and quantitative analysis of both ensemble and cell-by-cell data. Together, these methods enable high-throughput quantification of biomarker expression.

Cell isolation via spiral microfluidics and the secondary anchor targeted cell release system

“…Cells were washed, centrifuged at 500 g twice with 4 ml stain buffer, and resuspended in 300 μl stain buffer. The precision and accuracy of quantitative flow (qFlow) cytometry profiling has been rigorously tested . An LSR Fortessa (BD) was used for cell data acquisition; BD FACSDIVA software was used for data processing; and FlowJo (TreeStar) software was used for data analysis.…”

“…Developing diagnostic tools that enable accurate measurement of plasma membrane receptors, or “biomarker preserving diagnostics” is a necessary step toward precision medicine . Indeed, several cancers are marked by overexpression of receptors, second messaging signaling molecules, and other dysregulated biomarkers, so accurate measurements of these biomarkers would be effective for diagnostics.…”

“…Many lab‐on‐a‐chip approaches offer faster cell isolation . However, these approaches often involve physical or chemical methods of manipulating cells, which may cause irreversible cell damage to plasma membrane proteins and signaling pathways . Therefore, there is a need for more rapid cell isolation techniques that preserve the physiological expression of proteins and genes on and within the cells …”

Single-Cell Receptor Quantification of an In Vitro Coculture Angiogenesis Model Reveals VEGFR, NRP1, Tie2, and PDGFR Regulation and Endothelial Heterogeneity

“…Minced tissues were then incubated with 0.2% collagenase IV for 5 min at 4 • C. After the incubation, the collagenase-treated tissues were vortexed for 3-5 min with 1-min interval to achieve optimal tissue dissociation. Dissociated tissues were then added bovine serum albumin (BSA)-supplemented stain buffer [32], and filtered through cell strainers (size 70 µm) to ensure single-cell suspension. Cell suspensions were spun down at 500 ×g for 5 min, resuspended at 4 × 10 6 cells/mL in stain buffer, and then kept on ice until antibody staining.…”

“…We have previously shown that quantification of plasma membrane RTKs, including NRP1 and PDGFRβ, can be affected by enzymatic cell dissociation solution, such as TrypLE TM (Thermo Fisher Scientific, MA, USA) [5,32]. To assess the effect of the coculture dissociation protocol, which includes a 5-min incubation with collagenase IV and some rigorous mixing, we dissociated HUVEC and HDF monocultures using two methods: (1) The traditional method where HUVECs and HDFs monocultures were lifted and dissociated using a nonenzymatic dissociation buffer, CellStripper TM (Corning, NY, USA), as described previously [8,32]; (2) The coculture dissociation mimicry where CellStripper TM -lifted cells were incubated with HBSS with 2 mM EDTA (VWR, Chicago, IL, USA) and 0.2% BSA for 2-3 min, then with 0.2% collagenase IV for 5 min at 4 • C, vortexed for 3-5 min with 1-min interval, filtered through 70-µm cell strainers (Fisher Scientific, Hanover Park, IL, USA ) and resuspended in ice-cold stain buffer at 4 × 10 6 cells/mL. qFlow cytometry analysis showed insignificant differences of monoculture RTK concentrations between the coculture dissociation method and the traditional method ( Figure S2).…”