The more care you take when preparing samples for flow cytometry, the more likely you are to produce reliable results. But with different sample types requiring different approaches to preserve cellular integrity, it can sometimes be hard to know where to begin. This article highlights some common sample-prep challenges and suggests ways of overcoming them to improve the quality of your flow cytometry data.

Sample-prep challenges

The inherent single-cell nature of flow cytometry means that sample prep presents unique challenges. Not only must researchers maintain cell viability and prevent samples from aggregating, but they must also preserve the optimal ratio of cells to antibodies for immunostaining to be accurate and reproducible. Failure to achieve any of these objectives can lead to irreplaceable sample material being lost as well as wasting time and resources.

“Samples analyzed by flow cytometry are incredibly diverse,” reports Johannes Fleischer, group leader for marketing campaign management at Miltenyi Biotec. “They include cultured cells treated with potential small molecule drug candidates, blood samples collected during large cohort studies, and tissue biopsies excised during surgery. Oftentimes, sample material needs to be preserved before being further processed, and a primary challenge lies in identifying a storage method that helps standardize results by preserving the cells in their natural state.”

Where samples will be analyzed straight away, the length of time between harvest and immunostaining can be critical. “Excessive time between sample collection and immunostaining can lead to poor viability or the loss of fragile cells such as granulocytes,” explains Mike Blundell, Ph.D., field marketing specialist at Bio-Rad. This point is echoed by Claude Chew, senior flow cytometry core specialist at the University of Virginia, who notes that while cell viability can be maximized through improved cell detachment, dissociation, tissue digestion, and red blood cell (RBC) removal—depending on the nature of the sample—such steps must be carefully timed to ensure they are not over or under done. “Under digestion during tissue dissociation results in not sufficiently liberating cells of interest,” he says. “In contrast, over incubation with RBC lysis buffers can cause complete destruction of cells in the sample.”

According to Torsten Schultz-Larsen, Ph.D., field application scientist at ChemoMetec, ensuring cellular properties do not change during sample prep can also be problematic. “Resuspending cultured cells in PBS—a buffer that bears little resemblance to the environment in which the cells were grown—is common practice, especially when performing cell counts,” he says. “Yet a study has revealed PBS to have an adverse effect on cell viability when cells are counted using an automated cell counter. This is thought to be attributable to the shear stress introduced by many automated counting systems and adds yet another variable to a flow cytometry experiment.”

Tips for flow cytometry sample prep

So how can you tackle these flow cytometry sample prep challenges? Andy Jones, market development manager at DeNovix, points out that a broad range of reagents and instrumentation has been developed to help researchers standardize their approach and highlights the value of collaboration between manufacturers like DeNovix and flow cytometry experts such as Chew. His insights are included among the following tips suggested by all of the contributors to this editorial.

  • Where sample storage is required prior to analysis, consider using a specialized storage reagent for short-term preservation of samples at 4 oC rather than freezing; material can subsequently be archived at -80 oC
  • Select a suitable method for your sample type
    • Consult scientific literature and review conditions determined by other researchers; the Worthington Tissue Dissociation Guide is an extremely useful resource
    • Be aware that certain epitopes may be cleaved by enzymes
    • When working with a rare cell type, choose gentler methods where possible (e.g. try using Dispase® rather than trypsin to isolate iPSCs) and consider pooling samples or employing methods for cell enrichment
  • Rather than scraping to detach cells from culture dishes, use enzymatic methods such as trypsin or Accutase® or non-enzymatic methods such as EDTA  
  • Use wide-bore tips when pipetting samples to avoid harmful shear forces
  • Unless required for analysis, remove RBCs where possible; these can overwhelm instrument electronics and act as a ‘protein sink’ to sop up antibodies and other reagents
    • if using density gradient separation (e.g. Ficoll or Lymphoprep™) to remove RBCs, always ensure the centrifugation step is performed without the brake and at room temperature, and remember that this method may be unsuitable for avian or fetal samples that contain nucleated RBCs
    • if lyzing RBCs (using water, ammonium chloride, or a specialized solution such as Erythrolyse), always optimize the incubation time to avoid inadvertently lyzing leukocytes
    • where physical removal of RBCs is impossible, consider using CD45 to gate out RBCs (RBCs are negative for CD45 while leukocytes are positive) and set a FSC threshold during analysis above which only leukocytes will be detected
  • Minimize aggregation by using nucleases or chelators during dissociation/resuspension; adding DNase I at 25–50 µg/mL or EDTA up to 5 mM reduces clump formation
  • Filter samples through a cell strainer to remove aggregates
  • Ensure samples are thoroughly mixed before introducing them to the flow cytometer
  • When washing samples, avoid unnecessarily harsh conditions such as vortexing that could generate artefacts; centrifuge only as fast as required and never leave samples in the centrifuge; when aspirating media, do not remove all the supernatant as cells can die in a dry pellet
  • Use a viability dye to ensure cell counts are accurate
  • Consider using an automated cell counter rather than a hemocytometer to define standard counting parameters and eliminate operator bias
    • For cell cultures with little debris and high viability, staining with Trypan Blue and using brightfield illumination can yield accurate and reproducible results
    • For primary cells, samples with cell debris, low viability samples or small cells, fluorescence methods such as Acridine Orange/Propidium Iodide or Acridine Orange/DAPI are recommended; automated cell counters are available that allow switching between brightfield and single or dual fluorescence applications (e.g. ChemoMetec’s NucleoCounter® NC-202™ or the DeNovix CellDrop™ FL)
    • When working with highly aggregated cell types, an automated cell counter able to handle clumpy cells can be advantageous (e.g. ChemoMetec’s NucleoCounter® NC-3000™)
    • Where possible, count cells in an environment that resembles their origin (e.g. using culture media rather than PBS)
  • Before proceeding with immunostaining, perform a quick visual inspection using a microscope to check the quality and quantity of your sample
  • Thoroughly optimize your sample-prep method, paying particular attention to incubation times and temperatures, and reagent concentrations
  • Always standardize conditions between experiments to ensure consistent, reliable results