A missing sample prep tool for single-cell biology is found

Gentle and semi-automated Laminar Wash™ technology removes background, debris, doublets, and dead time, extending single-cell ‘omics budgets.

Single-cell ‘omics technologies are achieving a new paradigm in biology. As Eadweard Muybridge applied photography to settle the “unsupported transit” controversy surrounding the 19th century galloping horse, immunologists are utilizing single-cell transcriptomics (scRNA) to tease apart the heterogeneity of immune cell function and unlock new therapies and diagnostics. Combining scRNA with other measurements adds additional dimensionality to the data, enabling further insights (a.k.a., “multiomics”).

Muybridge horse paradigm shift

The Horse in Motion, the first instance of time-lapse photography, by Eadweard Muybridge (Library of Congress)

The scRNA multiverse

CITE-Seq, which adds barcoded antibodies that are simultaneously decoded by sequencing, enables immunophenotyping and transcriptomes to be read out at single-cell resolution from the same sample. Such multimodal analysis is being used to unravel the complexities of human disease, drawing detailed maps of myeloid cells in brain cancer, revealing novel pathways of immune checkpoint inhibitor resistance, and proposing an immune dysfunctional signature that distinguishes COVID-19 post-exposure recovery from fatality.

To maximize return on investment for individual studies, multiplexing techniques have been developed that allow assorted samples to be acquired at once, cutting down on costs and increasing statistical power. Such “hashtagging” kits like TotalSeq from BioLegend and CellPlex from 10X Genomics contain unique labels so that individual samples pooled together for acquisition can be identified upon analysis.

What multiomics and multiplexing methods provide in dimensionality and cost savings, they also add to sample prep complexity. Removing the additional reagents to suppress background signal necessitates equivalencies in cell or nuclei washing steps. For example, the CellPlex cell surface protein and multiplexing protocol calls for five washes prior to moving onto additional prep prior to acquisition, and provides additional guidance on cell or nuclei pellet handling. Each spin and resuspension represents an inefficient in-opportunity to lose additional viable precious cells or leave excess supernatant, particularly in samples where starting cell counts are low or target cells are known to be fragile, such as those taken from the tumor microenvironment. Perhaps that’s why Biolegend suggests using Laminar Wash systems as an alternative to centrifuge-based washing.

Laminar Wash technology represents a novel approach to preparing cell and nuclei suspensions for single-cell sequencing studies. Through gentle removal of unbound antibody, reagents, debris, and dead cells without centrifugation, the method often yields superior cell retention, especially for samples with low starting cell counts. This extensive washing procedure provides researchers with low background to strengthen data readouts, parameters to optimize preparation, including flow rates and number of wash cycles, as well as semi-automated walkaway capability to streamline workflows. Furthermore, single-cell leader and therapeutics company ImmunAI reported increased retention of single cells and fewer doublets in a 2020 webinar. Is Laminar Wash the missing sample prep tool to further unlock the promises of single-cell biology?

Proof is in the Publication

The COVID-19 pandemic is sparking what could be called a renaissance in B cell biology, for instance how memory B cells respond to mRNA vaccines in naïve and SARS-CoV-2 recovered individuals. Given the growing popularity of datasets at single-cell resolution, a team from University of Surrey and Kings College London surmised that extending the peripheral B cell diversity understood from phenotypic mapping to the transcriptome would represent a valuable resource for researchers. Since B cells comprise a minority of PBMCs, lead author Alex Stewart, Ph.D. pointed out that single-cell studies tend to “clump B cells together and analyze them as a single group,” missing out on their now-trendy immunology. The study, published in Frontiers in Immunology, aimed “to give researchers a better idea of what specific cells they were looking at, rather than just calling them all B cells,” he explained.

The team used FACS sorting to isolate the five canonical peripheral B cell subsets, including the esoteric CD19+CD27-IgD- “double negative”, and proceeded to collect single-cell transcriptomics data, running the resulting five samples each on a lane of a 10X Genomics Chromium 10X instrument. Cognizant of needing confirmatory donor samples and a limited sequencing budget, they collected peripheral B cells from two additional donors in a similar manner. Only this time, TotalSeq was employed to individually label and pool each of the resulting 10 samples on one 10X lane, and a Laminar Wash MINI system was used to prepare the samples, rather than centrifugation. In effect, they maximized their time and budget to get the data they needed (see image).

Laminar Wash method is the superior sample prep choice for cell hashing studies

Single-cell transcriptomics and FACS phenotypic sorting data. The researchers used Laminar Wash MINI to process the five FACS-sorted B cell subpopulations from two additional donors (10 total samples) and subsequently analyze on a Chromium 10X controller (Adopted from Stewart, et al. (2021) Front Immunol.).

“That’s where the Curiox comes in, because it’s better at cleaning unbound antibodies when you do cell hashing, so you get less background when you run samples through the 10X,” says Dr. Stewart. He commended Laminar Wash’s ability to retain cells, particularly for samples expected to contain low starting numbers, which centrifugation definitively chips away at with each additional round. Not to mention skipping so many spins “allows you to minimize your workflow, and make yourself more efficient,” he added.

“[Laminar Wash] made life a bit easier, saving us time, effort, and money.”-Alex Stewart, Ph.D., University of Surrey

The resulting dataset revealed developmental trajectories of B cells with ten distinct clusters, including a novel double-negative population expressing IgE antibodies. In essence, the work provides a baseline landscape to evaluate shifts in B cell subpopulations, such as blood from COVID-infected individuals. Remarkably, the TotalSeq hashed, Laminar Wash-processed samples recapitulated the primary map, despite ~1/10 the number of starting cells analyzed per donor. Overall, the individual landscapes speak to a similar structure with underlying uniqueness attributable to B-cell biology.

In further characterizing the effects of ageing and infection status on B cell-mediated immune responses, Dr. Stewart foresees the lab extending their single cell efforts, leveraging their new Laminar Wash HT2000 better suited for higher throughput applications in sample multiplexing and retaining low numbers of precious cells.

What could you do with the extra time and research dollars a Laminar Wash system could save your lab? We’d be happy to demonstrate its capability with your protocol, just let us know.

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