Advances in Single-Cell Analysis

Single-cell analysis encompasses an entire workflow, starting with cell isolation (which may include flow cytometry, laser capture microdissection, microfluidics, or levitation), perhaps followed by some type of nucleic acid-based assay for genomic, transcriptomic, or epigenomic analysis. Single-cell analysis is the only way to distinguish the varieties of responses that make up those of a cell population, whether cells grown in a lab, cells comprising a tumor, or the complex immune cell responses that fight that tumor. “Single-cell analysis tools provide critical insights across various fields and enable discoveries previously impossible with bulk population-level analysis,” says Angelica Olcott, Market Development Manager at Bio-Rad Laboratories. “They are central to multiomics research, impacting areas like cancer, immunology, neuroscience, and developmental biology.” This article looks at new tools used to speed up the discovery process by optimizing single-cell workflows.

Cell profiling and enrichment

An efficient means of analyzing high volumes of single cells in solution is flow cytometry, which flows individual cells past a laser beam and measures the scattering or absorption of light and whether each cell is tagged by fluorescent labels. This high-throughput method is especially valuable for profiling immune cells, for example, which can be categorized by their expressed cell surface markers. “Flow cytometry can dissect immune responses by identifying key immune cell types involved in infections or autoimmune diseases,” says Olcott. Bio-Rad’s ZE5 Flow Cytometer, with 5 lasers and 30 detection parameters, is well-equipped to analyze multiple biomarkers simultaneously. For compatible fluorescent markers, “the stability and high intensity of StarBright Dyes ensure reliable detection of low-abundance targets across multiple applications, including flow cytometry, imaging, and western blotting,” says Olcott.

Bio-Rad offers tools for cell enrichment, which can be linked with downstream single-cell analysis for a complete workflow. For example, the Genesis Celselect enriches live cells from blood for staining, counting, or single-cell RNA sequencing. “When Genesis Celselect’s cellular enrichment capabilities are integrated with the 3' Single Cell RNA Sequencing Kit, it can potentially support more detailed gene expression studies involving isolation of individual cells for RNA sequencing,” says Olcott. “Single-cell RNA sequencing (scRNA-seq) is an ideal technique for uncovering tumor heterogeneity and distinguishing rare subpopulations, such as drug-resistant cancer cells.” Bio-Rad provides another cell enrichment tool, the ddSEQ Isolator, which is compatible with their single-cell ATAC-Seq kit. “Single-cell ATAC-seq reveals epigenetic changes, and is a useful approach to delineate embryonic development, and monitor how stem cells differentiate,” adds Olcott.

Workflow diversity

As in many research areas, single-cell workflows have grown increasingly diverse by incorporating more types of techniques than ever before. Iván Godinez, Director of Product at Proteintech Genomics, notes growing interest in single-cell multiomics, especially “measuring proteins via the use of DNA-barcoded antibodies along with whole-transcriptome profiling,” he says. “By adding high-plex protein measurements to scRNAseq experiments, researchers are obtaining a more holistic understanding of cell function, cell diversity, and how cells change in response to therapeutics.” Proteintech Genomics will soon release the MultiPro^® Human Discovery Panel for profiling 325 unique proteins via 346 DNA-barcoded antibodies. “The Discovery Panel represents a leap forward in single-cell multiomics, providing researchers with the ability to characterize transcription factors, cytokines, signaling proteins, phospho-epitopes, and much more,” says Godinez.

Godinez believes that single-cell analysis contributes a valuable, high-level view of the biology of cells. “With the addition of high-plex single-cell protein and RNA multiomics, researchers can achieve a significantly improved understanding of single-cell biology, including [in] immunology, oncology, drug development, and developmental biology, to name a few,” he says. For example, researchers using CRISPR to knock down gene expression in cell populations (perhaps during studies of drug targets) can use Proteintech’s platform to verify these effects at the single-cell level. “By using the high plex MultiPro^® Human Discovery Panel, researchers can not only understand how gene expression changes during CRISPR screening, but also understand how proteins are impacted,” says Godinez.

Functional assays

The downside of studying cells at the population-level is that varied cellular responses may be measured as an average, and then mistaken for the biological response at the single-cell level. Lightcast supports researchers who are seeking to assay single-cell function directly. “We believe that a core capability missing from many of today’s single-cell technologies is the ability to directly measure functional behavior of individual cells,” says Paul Steinberg, Chief Commercial Officer at Lightcast. “Current technologies allow researchers to create powerful datasets from which they can infer functional relationships, but none has been able to directly measure such relationships in real time and at scale.” Lightcast’s approach uses microfluidic chips with transparent sides that allow beams of light to control the movement of individual droplets containing single cells. Within a chip, you can construct massively parallel droplet arrays to study single cells in a high-throughput and flexible format.

Lightcast’s platform also offers scientists the ability to subdivide and sort a mixed cell population by assaying according to different criteria, such as expression of surface markers or ability to bind different antibodies. Such protocols may require transferring to other platforms for additional assays, incurring risks of time loss, cell loss, and contamination. “At Lightcast, we have built sequential assays directly into our single-cell functional analysis system, which is currently available through an early access program,” says Steinberg. “Scientists can sort and re-sort cells into subpopulations and assay them as needed, and because cells do not have to leave our platform, they remain viable for much longer.”

The increasing application of single-cell analysis to a variety of cellular functions and dysfunctions may shed light on previously misunderstood cell-based responses. Furthermore, the ability to incorporate data from genomic, transcriptomic, and epigenomic modalities provides today’s researchers an unprecedented view of single cells as busy purveyors of complex information that can deeply impact human life.