When it comes to high speed analysis of large, heterogeneous cell populations, flow cytometry is hard to beat. Yet while many researchers invest considerable time and effort optimizing antibody staining protocols, the importance of upstream sample preparation should never be overlooked. Irrespective of the sample type under investigation, problems such as streaking, weak or no fluorescence, and loss of epitope can begin at sample collection and worsen all the way through fixation and permeabilization. By ensuring sample preparation techniques suit the cell type and marker being evaluated, these issues can be avoided.
Pay attention to culture conditions
“If you’re working with cultured cell lines, many different factors can impact on flow cytometry results,” notes Julie Clor, senior scientist at Luminex Corporation. “These include the culture media, supplemental proteins, and confluencies, as well as the stimulation or treatment conditions used to promote expression of the antigen of interest. Keeping cells in log phase growth will help prevent cell death and potential clumping, while using EDTA or enzymes such as Accutase® to maintain single cell suspensions is an effective way to rule out aggregates. It is also sensible to avoid harsh centrifugations as these can damage cells and exacerbate the formation of clumps. By handling cells appropriately both prior to and during sample collection, flow cytometry data will be far more robust.”
“One way of ensuring treatment conditions are consistent between experiments is to use pre-formulated reagents,” explains Fabrice Malergue, senior staff development scientist R&D research at Beckman Coulter Life Sciences. “For instance, it is now possible to obtain optimized cocktails of common activators such as PMA-ionomycin or LPS +/- Brefeldin A that have been unitized in flow cytometry tubes and dried to guarantee room temperature stability. Adding whole blood or cells directly to these tubes eliminates the variability associated with preparing activators manually, helping to improve experimental reproducibility.”
Always check cell concentrations
Although initial cell counts are often fairly predictable when harvesting samples for an experiment that has been run multiple times before, event rate should always be checked at the point of collection to prevent fluctuating cell concentrations affecting results. “Running your sample with too high of a cell concentration can cause an increase in electronic abort rates, thereby losing valuable events from your analysis,” reports Lauren Jachimowicz, Ph.D., applications development scientist at Agilent Technologies. “However, decreasing your cell concentration can increase the total volume necessary for a statistically relevant number of events to be collected, thereby extending your run time. Determining an optimal cell concentration range for your experiment and ensuring you stick to it will help preserve the integrity of your data.”
Smita Ghanekar, Ph.D., associate director, medical and scientific affairs at BD, notes that a further complication associated with running more concentrated samples is that cells are more likely to aggregate and clog the flow cytometer during acquisition, invariably causing lengthy delays to experimental timelines. Clor adds that high cell concentrations can also lead to the appearance of doublets or false positives and can influence antibody and dye uptake as this depends on the cell-to-marker ratio. “Despite there being many variables that can affect flow cytometry data, the potential impact of sample preparation methods can easily be evaluated by measuring the cell concentration in parallel with viability at various stages of the process,” says Clor. “This can rapidly be accomplished using a product such as Guava® ViaCount™ Reagent or Muse® Count & Viability. Since unexpected concentrations or low viabilities can cause erroneous results, checking these parameters can save both time and precious reagents.”
Image: Absolute total cell counts and viability of dead and dying cells using the Muse® Cell Count and Viability Kit. Image courtesy of Luminex.
Understand the fixation process
While it may be tempting to view sample preparation as complete once cells have been fixed, there is certainly no room for complacency; fixation must be carefully optimized just like any other stage of a flow cytometry experiment. Malergue highlights as an example that formaldehyde and paraformaldehyde—both commonly used reagents to fix samples for flow cytometry—are not equivalent in strength and consequently react very differently at the same apparent concentration. “Researchers should always optimize the final fixative concentration alongside key parameters such as fixation temperature and time to produce consistent results,” he says. “Furthermore, it is important to remember that fixatives act extremely rapidly and should be mixed immediately when added to samples; failure to do this will mean the sample is exposed to a nonuniform fixative solution that can cause some regions to become highly fixed and leave other regions not fixed enough.”
Ghanekar explains that fixing for too long or using an overly harsh fixative for the sample type can also be problematic, often causing epitopes (pre-staining) and/or fluorochrome tags (post-staining) to be compromised. “Inadequate washing after fixation can exacerbate this issue by allowing any fixative remaining in the medium to continue acting,” she says. Jachimowicz adds that because fixative-induced alterations to antigens can lead to weak or nonexistent surface staining, it is recommended that researchers stain for surface antigens before fixing samples, only proceeding to intracellular staining once this stage of the protocol is complete.
Strike the right balance between fixation and permeabilization
In addition to the effects already described, cell concentration can also influence the efficiency of permeabilization since it typically relies on detergents that target membrane lipids. “Proper permeabilization is critical for epitopes to be accessible to antibody reagents,” says Malergue. “If a permeabilization is too strong it can cause epitopes to be lost, however an insufficient permeabilization will not support intracellular staining. It should be understood that there is a balance here: a strong fixation can allow a strong permeabilization, but although this procedure is more robust, it requires more washes and can be detrimental to cell shape, number, and some epitopes; a weak fixation can be advantageous, but must be associated with a mild permeabilization. Whichever route you choose, both steps must be tightly controlled for data to be reproducible.”
Every step counts
Whether the aim of a flow cytometry experiment is to investigate cultured cells, primary cells, blood, PBMCs, or dissociated tissue samples, the success of any study begins with proper sample preparation. To minimize the occurrence of problems, not only at this stage but also further downstream, researchers must optimize protocols from start to finish; this can easily be achieved by leveraging the wealth of expertise and enabling products available.