A core laboratory technique used to characterize and sort highly complex cell populations, flow cytometry has widespread utility. Benefiting from the potential to detect greater than 20 parameters within a single sample, the popularity of flow has grown alongside its increasing capacity for data generation. Yet while simultaneous analysis of multiple parameters is hugely informative, it’s important to remember that flow cytometry data will only be relevant if due care is taken during sample preparation. This means handling samples appropriately before embarking on often lengthy immunostaining protocols. By avoiding key mistakes from the outset, researchers can have greater confidence in experimental results.
1. Effective sample preparation begins at the point of collection
Samples destined for flow cytometric analysis are incredibly diverse, ranging from cultured cells, to blood, to tissue biopsies. These require very different approaches to sample collection and processing. “Blood or tissue samples are often collected days, weeks, or even months prior to analysis, such as in large cohort studies running in a centralized facility,” explains Johannes Fleischer, global product manager at Miltenyi Biotec, “yet poor storage and preservation can compromise results. To attain a high degree of standardization, it’s worth considering specialized tissue storage reagents, freeze-drying, or preservation at -80oC to reduce experimental variability.”
2. Consider the need for cell enrichment
“Often, researchers use flow cytometry to evaluate the activation status of cells upon treatment with a stimulus such as an antibody or cytokine,” reports Ekaterina Zvezdova, technical service scientist at BioLegend. “However, antibody or cytokine-mediated stimulation of cells often results in internalization of surface receptors. For example, anti-CD3 antibody stimulation of T cells causes down-regulation of the TCR/CD3 complex on the T-lymphocyte surface, resulting in failure to use CD3 or TCR molecules to identify T cells within a heterogeneous population. By anticipating the effects of different treatments on specific cell markers, researchers can implement effective strategies to gather data. Approaches include the use of alternative lineage markers for cell identification or enriching the population of interest before stimulation.”
Also highlighting the benefits of cell enrichment, Fleischer explains that rare cell types such as circulating tumor cells, hematopoietic stem cells, and antigen-specific T cells can be especially hard to capture in a flow cytometry experiment. “Using magnetic beads conjugated to antibodies, it’s possible to enrich rare cells within a magnetic column,” he says. Our MACS® microbead technology represents the gold standard approach to this method, and by integrating a magnetic enrichment column into our MACSQuant Analyzer flow cytometers, we’ve made it possible to enrich cells within the instrument automatically. These can then be analyzed straight away.”
3. Dissociation can affect antibody binding
It’s well-known that flow cytometry requires single cells in suspension, therefore when researchers wish to analyze cells derived from solid tissues, samples must first be dissociated. Mechanic disruption and enzymatic digestion are the usual methods chosen to achieve this, yet both cause cellular stress and can lead to poor viability. “To improve reproducibility through better mechanical tissue dissociation, we developed our GentleMACS™ dissociator—an automated instrument featuring pre-set programs to more gently dissociate specific biological materials,” explains Fleischer. “To complement the GentleMACS we’ve designed a range of tissue-specific dissociation buffers. We’ve generated extensive testing data for these, demonstrating that they optimally preserve surface epitopes.”
“Enzymatic digestion can often affect the antibody binding epitope,” notes Zvezdova. “For example, trypsin/EDTA treatment of adherent cells is known to result in cleavage and inactivation of cadherins, making it difficult for anti-cadherin antibodies to recognize their target. To mitigate these effects, it can be wise to use a gentler detachment solution such as Accutase®.”
Whichever form of dissociation is used, Fleischer suggests that cells should subsequently be filtered through a strainer to remove any clumps from the sample. “Although they’re just routine laboratory consumables, strainers are key to preventing blockages and sample overflow—a painful moment most researchers have encountered at some stage during a flow cytometry experiment,” he says.
4. Fixation also requires optimization
Fixation is another sample-preparation step that can affect the antibody binding epitope, with some antibodies demonstrating a loss of signal when used to stain fixed cells. “We’ve performed in-house testing for some of our antibody clones, comparing unfixed cells and cells fixed with 4% paraformaldehyde prior to staining,” reports Zvezdova. “These results provide guidance regarding the likely success of staining a fixed epitope and, if information on the clone of interest is not provided, we generally recommend performing surface staining before fixing the cells.”
Image: Epitope alteration may cause some antibodies to demonstrate a loss of signal when used to stain fixed cells. Image courtesy of BioLegend.
Temperature can also impact significantly on antibody binding, for example leading to rapid internalization of surface proteins such as chemokine and cytokine receptors. If cells are to be immunostained prior to fixation, it can be beneficial to perform antibody incubations at room temperature or 37oC, rather than at 4oC.
5. Fc blocking can help to minimize background
A major source of unwanted background staining, antibody binding to Fc receptors on immune cells can yield false positive results or mask the presence of low-abundance targets. It is therefore sensible to consider including an Fc blocking step before progressing to immunostaining. “While Fc blocking reagents can be employed to minimize background staining, their use adds an extra step into protocols and can result in loss of cells,” notes Fleischer. “To remove the need for Fc blocking, we developed REAfinity™ recombinant antibodies. These benefit from an engineered Fc region, which prevents binding to Fc receptors and yields more consistent flow cytometry data.”
6. Viability stains can inform sample preparation technique
“It can be sensible to perform a cell count at multiple stages of the sample-preparation process to confirm significant cell loss hasn’t occurred,” notes Catherine Klapholz, research scientist at Nanna Therapeutics. “The inclusion of a viability stain can provide useful information here, informing the impact of different sample-preparation techniques on the overall cell population. It is, however, important to test the stability of this staining. If the assay follows the intensity of a signal, researchers should check that the geometric mean or the median signal of one tube is the same at the end of the acquisition as it is at the beginning. Acquisition can sometimes take 1–2 hours, and left at room temperature or even at 4oC, the signal can dramatically increase.”
No matter how thoroughly the sample-preparation process is optimized, it should of course never be assumed that a protocol that works well for one cell line will work equally well for another. By tailoring sample-preparation protocols carefully to account for experimental differences, researchers stand a far greater chance of achieving flow cytometry success.