Flow cytometry is the classic technique for single-cell analysis, providing valuable insight into the presence and abundance of different cell types within a heterogeneous population. In recent years, flow cytometry experiments have become increasingly complex, yet one factor remains indisputable: the success of any flow cytometry experiment hinges on the preparation of single-cell suspensions with high viability, well-preserved cell surface antigens, and a low incidence of aggregates or cell debris. This article describes effective ways of achieving single-cell suspensions and touches on approaches to confirm that these are of a suitable quality to deliver accurate and reliable flow cytometry results.
Poor sample preparation compromises data quality
Inadequate preparation of single-cell suspensions for flow cytometry can cause many different problems. While cell clumps can clog the flow cytometer and delay workflows, smaller cell aggregates can skew data by generating false positive results. Low viability not only limits the number of cells available for investigation but also results in large amounts of cellular debris that can lead to autofluorescence and unwanted background signal, often causing rare events to be lost. Additionally, cellular epitopes are easily damaged if enzymes are used inappropriately. Taking the time to address these issues promises to improve data quality.
“A researcher will often have a better chance of achieving a quality single-cell suspension if they start with a sample that is healthy and viable,” reports Julie Clor, senior scientist at Luminex. “For cultured cells, it is important to ensure the cells are not overgrown and that proper media and supplements are utilized. During preparation steps, especially for adherent cultures, close attention should be paid to timing when using dissociation reagents or enzymes so they do not damage the cell membrane or surface proteins. Cells should always be handled gently, without subjection to harsh vortexing or centrifugation forces, and it is critical to understand how long cells can be left on ice, as well as the impact of any freeze-thaw cycles.”
Image: A quality single-cell suspension will have high viability, well preserved cell surface antigens, and a low incidence of aggregates or cell debris. Image provided by Agilent Technologies.
Jana Soyka, global product manager for sample preparation, cancer research, and MACSmolecular at Miltenyi Biotec, notes that freezing is often unavoidable, especially when working with biopsy material gathered during large cohort studies. However, specialized tissue storage solutions are now available that eliminate the need to process freshly excised tissue straight away, making it easier for researchers to collect samples from multiple origins or at different time points. “Storage of up to 48 hours should ideally be carried out in a cell or tissue storage solution at 4oC rather than freezing,” she says. “Where freezing is inevitable, freezing protocols should always be chosen carefully to preserve the natural state of the cells and prevent necrosis or apoptosis.”
Cell source dictates sample-preparation methods
The process for preparing a single-cell suspension is largely determined by the origin of the cells. Lauren Jachimowicz, application development scientist, flow cytometry at Agilent Technologies, notes that cultured cell lines can readily be transformed into a single-cell suspension either by gentle agitation with a pipette to break up small clumps of suspension cells and dissociate loosely adherent cells, or by dislocation from the culture plate or flask using an appropriate dissociation buffer. In most laboratories, trypsin is the go-to reagent to harvest adherent cells, however in some cases a gentler reagent, or even an enzyme-free dissociation buffer, may be a better choice to avoid damaging cell surface epitopes.
Working with whole blood requires a different approach, depending to some extent on the aim of the experiment. “Lysis of red blood cells is often sufficient to analyze leukocytes by flow cytometry,” notes Dr. Alexandra Wittmann, senior scientist at Abcam. “However, if mononuclear cells are preferred, they can be isolated using density gradient centrifugation with media such as Ficoll-Paque.” Clor adds that whichever cellular types are being interrogated within a whole blood sample, proper blood collection practices are essential. “The storage temperature, adequate and appropriate anti-coagulant in the blood collection tubes, age of the blood, and appropriate mixing can all help to prevent clotting or clumping of the cells and non-specific interactions,” she says.
Solid tissues are particularly challenging to work with, usually requiring a combination of mechanical and enzymatic dissociation to produce a viable single-cell suspension. “In tissues, cell types are embedded to different degrees,” explains Soyka, “meaning that carefully selected enzyme combinations are required to release all cell types and to reflect the true heterogeneity of the sample. For instance, when working with brain tissue, isolating fragile neurons necessitates a different method than isolating immune cells. Protocols must be designed in a way that considers not only the nature of the tissue but also the nature of the cells of interest.”
Collagenase and DNase are among the most commonly used enzymes to prepare single-cell suspensions from tissue samples—collagenase to break down components of the extracellular matrix and DNase to digest free DNA that can cause aggregates—yet Wittmann highlights that care must be taken in collagenase selection as some collagenases have proteolytic activity that can damage the protein target of interest. “Using the right collagenase and DNase can certainly be beneficial,” she says. “For tissues like rodent spleen, mechanical dissociation might seem enough, but treatment with these enzymes can increase recovery of dendritic cells and macrophages.” Adding EDTA to buffers and using cell strainers to remove clumps can also enhance the quality of single-cell suspensions.
The value of confirming sample uniformity
Before analyzing cells by flow cytometry, it is a good idea to use reagents such as Guava® ViaCount™ or Muse® Count & Viability Reagent to confirm sample quality by monitoring absolute cell counts and viability. Following acquisition, the scatter channels can be useful to exclude coincident events known as doublets. “Doublets occur when two cells pass the laser simultaneously,” explains Wittmann. “This causes the signals from both to be detected as one event, generating false positives or appearing as double DNA content during a cell cycle analysis. Doublet exclusion can be achieved by plotting forward scatter height (FSC-H) against forward scatter area (FSC-A), whereby single cell events will appear as a diagonal while doublets appear higher in FSC-A but not in FSC-H.” Gating samples by plotting data as time against scatter allows for exclusion of poor data caused by an instable sample flow.
With a wide range of enabling products available to simplify the preparation of single-cell suspensions for flow cytometry, researchers are increasingly likely to achieve reliable data the first time, irrespective of sample type. Ready-to-use kits preserve epitopes during tissue dissociation; provide rapid removal of dead cells or debris; and allow for pre-enrichment of defined cell sub-populations; while modern flow cytometers are equipped with software features to assess sample quality and the ability to gate out dead cells and perform doublet discrimination. Systems like Guava® easyCyte™ HT also integrate paddle mixers for gentle yet effective sample pre-mixing immediately prior to acquisition. As further developments evolve, flow cytometry in single-cell analysis will continue to drive future scientific discovery.