Cell Surface Profiling Using High-Throughput Flow Cytometry: A Platform for Biomarker Discovery and Analysis of Cellular Heterogeneity

Abstract

Cell surface proteins have a wide range of biological functions, and are often used as lineage-specific markers. Antibodies that recognize cell surface antigens are widely used as research tools, diagnostic markers, and even therapeutic agents. The ability to obtain broad cell surface protein profiles would thus be of great value in a wide range of fields. There are however currently few available methods for high-throughput analysis of large numbers of cell surface proteins. We describe here a high-throughput flow cytometry (HT-FC) platform for rapid analysis of 363 cell surface antigens. Here we demonstrate that HT-FC provides reproducible results, and use the platform to identify cell surface antigens that are influenced by common cell preparation methods. We show that multiple populations within complex samples such as primary tumors can be simultaneously analyzed by co-staining of cells with lineage-specific antibodies, allowing unprecedented depth of analysis of heterogeneous cell populations. Furthermore, standard informatics methods can be used to visualize, cluster and downsample HT-FC data to reveal novel signatures and biomarkers. We show that the cell surface profile provides sufficient molecular information to classify samples from different cancers and tissue types into biologically relevant clusters using unsupervised hierarchical clustering. Finally, we describe the identification of a candidate lineage marker and its subsequent validation. In summary, HT-FC combines the advantages of a high-throughput screen with a detection method that is sensitive, quantitative, highly reproducible, and allows in-depth analysis of heterogeneous samples. The use of commercially available antibodies means that high quality reagents are immediately available for follow-up studies. HT-FC has a wide range of applications, including biomarker discovery, molecular classification of cancers, or identification of novel lineage specific or stem cell markers.

Identification of neutrophil surface marker changes in health and inflammation using high-throughput screening flow cytometry

“…All cells were resuspended in staining media containing eFluor506 or eFluor780 to identify non-vital cells. Fifty microliters of the above mixture was added directly to each well, preloaded with 2-5 μl of each individual antibody based on previous plate set up and validation [16] (for a full list of antibodies used, please see supplement Table S1) and incubated for 30 min in the dark. Plates were then centrifuged, and medium was removed by aspiration.…”

Immunophenotyping and Transcriptomic Outcomes in PDX-Derived TNBC Tissue

“…These studies show the benefit of this approach, offering more detailed characterization and analysis than with conventional bulk methods (7,23,47). Our study shows that a systematic flow cytometry survey with an 11-color panel resulted in a novel characterization tool capable of phenotypically profiling a range of breast cancer samples beyond the current standard (e.g., hormone receptor status).…”

“…Once identified, such subpopulations can be subjected to downstream analysis to elucidate cellular function (22). Some large-scale screening campaigns of cancer cell lines identified a range of potential surface markers that can be used in this fashion (23). More systematic tools and methods are urgently needed to make this approach accessible to research and clinical laboratories investigating solid tumor biopsies, and a more refined understanding of heterogeneity is necessary to positively affect patient care (24).…”

High Throughput Assay Identifies Glafenine as a Corrector for the Folding Defect in Corneal Dystrophy–Causing Mutants of SLC4A11

“…26,54 Other high throughput assays to detect protein at the cell surface have been developed. [66][67][68][69] Although there are other methods to measure protein abundance at the plasma membrane, including cell surface biotinylation, flow cytometry, or confocal microscopy based immunofluorescence, these are relatively time and material intensive. We developed a simpler assay, including elements of an assay developed to assess F508del CFTR cell-surface processing.…”