Flow cytometers perform a variety of multi-parametric applications and have been used for an expanding set of cell analysis applications over the past forty years.
Today’s life science researchers must utilize technologies from many disciplines, such as molecular biology, immunology, and cell biology, to advance their research. Accumulating, and then mastering varied technologies and instruments devours time as well as valuable funding resources. Often in research, a technique may be needed in only a certain experimental phase leading to an accumulation of infrequently used instruments cluttering valuable bench top space. The ideal circumstance would be to have an instrument, which could perform a variety of multi-parametric applications.
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Figure 1: 18 inches wide and 17 inches deep, (36.3 × 41.9 cm), the 30-pound (13.6 kg) Accuri C6 Flow Cytometer fits on any lab bench and features two lasers and six detectors. |
Multi-parametric flow cytometry
Flow cytometers are just that tool and have been used for an expanding set of cell analysis applications over the past forty years. This quantitative, multi-parametric technology is often used to study cellular phenomena that might also be investigated by fluorescence microscopy, imaging, or microplate reading. Such experiments include those designed to understand the cell cycle, apoptosis, cell proliferation, and viability as well as assessment of transfection efficiency, determination of cellular and molecular profiles, monitoring of cell cultures, and performance of protein multiplex bead assays.
However as with so many technologies, manufacturers have added a seemingly endless series of bells and whistles to mature flow cytometric platforms over time, rendering them increasingly unwieldy. Only trained, experienced operators can effectively use these systems and workloads can back up to the point where progress is gated by access to a core lab.
New-generation, compact flow cytometers, such as the Accuri C6 Flow Cytometer System (Figure 1), have simplified the flow cytometric process and offer several unique advantages over fluorescence microscopy, microplate reading, qPCR, and Western blots by enabling multi-parametric, individual-cell analysis. Pre-optimized detectors calibrated to operate within their linear range can be used to analyze a wide variety of samples, ranging from dim, barely-fluorescent, micron-sized platelets through large, >30 micron, highly-fluorescent cell lines. By incorporating a linear dynamic range greater than six decades, an Accuri C6 can quantitatively capture the entire scope of biological variations in a single run without the need for data acquisition optimization or tuning. Single to hundreds of thousands of individual cells, from heterogeneous populations, in hundreds of samples, can be analyzed and rare events flagged. Another powerful feature is the ability to quantitatively measure the concentration of cells or particles in samples. Absolute cell counts are correlated with the specific, known volumes being sampled.
Varied Biological Applications
We will now briefly examine how flow cytometry can improve outcomes from six existing techniques.
Western Blots
Western blots are considered a gold standard for protein biomarker determination. However in many cases, flow cytometry offers specific advantages over Western blots in characterizing protein expression. While Western blots represent expression of a cell population, flow cytometry characterizes individual cells, allowing further characterization of a specific cell population. This is particularly beneficial when attempting to characterize small subset cell populations. For example, characterizing the aberrant disease associated phenotypes of viable Minimal Residual Disease (MRD) cells in apoptosis studies is difficult to address using Western blots due to the small proportion of MRD cells in the total population. Flow cytometry allows one to selectively analyze MRD cells and is rapid and highly sensitive.
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Figure 2: Dot plots of continuous calcium dynamics, obtained on the Accuri C6, clearly indicate the absence of gaps on addition of test compounds. Fluorescence of Fluo-4 and Forward Scatter versus time.1 |
GFP Transfection Studies Flow cytometry can accurately quantitate reporter gene expression (such as, Green Fluorescent Protein, GFP) in each cell in a population being transfected. Co-transfection of a reporter plasmid and a reference plasmid can be quite variable in normal human cells, making interpretation difficult in reporter gene assays. However, using multi-parametric flow cytometry, reporter and reference plasmid expression can both be quantitated at the single cell level through the use of fluorochrome-conjugated antibodies to the transfected gene product. In addition propidium iodide can be used to monitor the DNA content of cells of varying viabilities, identifying apoptotic cells with sub G1 DNA content.
Continuous Measurement of Intracellular Ca2+ An alteration in intracellular calcium ions (Ca
2+) is one of the most rapid cellular responses to a variety of stimuli, yet obtaining accurate data on the dynamics of intracellular calcium is a major challenge for research in this field. Historically, specialized liquid handling and fluorescence microplate reading systems have been used to examine bulk population behavior of cells during investigations of the rapid responses of intracellular Ca
2+ to various stimuli
in vitro. However, the use of a flow cytometer, which operates with an open, as opposed to a pressurized, fluidics system allows continuous monitoring of cells upon the addition of test compounds, providing a method for highly accurate, dynamic calcium measurements. With conventional flow cytometry, the run has to be halted, the sample tube opened and agonist added, then the tube resealed to recommence data acquisition, which adds a gap, or blind spot, in the data (Figure 2).
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Figure 3: Accuri C6 flow cytometric analysis of homogenates prepared from Arabidopsis thaliana root tissues. A) Biparametric contour plot of FL2-A vs. FL3-A fluorescence emission. B) Enlargement of the square region-of-interest (R1) containing the nuclei. The nuclei are enclosed in the polygonal region P1. C) Uniparametric histogram of FL2-A fluorescence, gated on region P1 of Panel B. Abbreviations 2C, 4C, 8C, 16C, 32C designate the appropriate C-values for the individual peaks.2 |
Cell Cycle Analysis and Ploidy Plant nuclear DNA content (ploidy) varies over extreme ranges and quantitative measurement of the characteristic “C-value” is achieved by detecting propidium iodide fluorescence using flow cytometry. Nuclear DNA measurements are often hampered by excessive cellular and sub-cellular debris and autofluorescence from other prevalent cellular components, such as chloroplasts. With more than six decades of digital signal linearity, the Accuri C6 is the first flow cytometer to cover the full biological range of flowering plant genome sizes from 0.32 to 80.9 picograms in a single run.
2 For example, the tiny 157MB
Arabidopsis thaliana genome is characterized by a 2C value of 0.32 picograms and a cluster in the biparametric contour plot for the endoreduplication series of 2C, 4C, 8C, 16C, and 32C (Figure 3). Accuri C6 data shows a near-perfect linear correlation of the data with r2=0.9999 and %CVs for the first four peaks of about 3%.
RNAi Knockdown RNAi has proven to be a powerful, genome-wide technique for studying protein loss-of-function via gene knockdown. Short interfering RNAs (siRNAs) can be transfected into cell populations to effect gene silencing and libraries of known siRNAs have been created for a wide variety of gene families. To assess the reduction in gene expression, quantitative real-time PCR is often the method of choice to measure mRNA levels. However, down-regulation of mRNA does not always correlate well with corresponding protein levels, especially those with low turnover rates. While Western blots can be used to quantitate these proteins, the dynamic range of detection is narrow. Both qPCR and Western Blots require bulk cell lysis and the resulting mixture of all mRNAs and proteins makes results from these approaches only valid for samples with uniform cell populations.
Two situations of 16% overall knockdown would be indistinguishable with qPCR or Western blots: 1) 80% of cells in a subpopulation that experienced 20% down-regulation per cell and 2) 20% of cells in a subpopulation that experienced an 80% down-regulation per cell. In practice, siRNA transfection efficiencies can be low and variable, so a better approach is flow cytometric measurement of intracellular protein knockdown in situ with viable intact cells from all subpopulations.3 Since flow cytometry is multi-parametric, fluorescent antibodies can be used to not only assess the siRNA-targeted protein levels, but also to detect other functional cell surface marker signals in the same cell. Various subpopulations in a heterogeneous sample can be quantitatively analyzed without the use of potentially function-altering cell purification protocols.
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Figure 4: The average coefficient of variation for replicate cell counts using three different counting methods on the same samples. A paired student’s T test was used to determine p values (95% confidence, N = 23). The direct-volume measurement method provides the least variability between replicate cell counts (average CV values: direct-volume measurement performed on the Accuri C6 = 2.10%; counting bead method = 2.94%; hemacytometer = 19.51%).4 |
Cell Counting The ability to quickly and precisely determine the absolute number or concentration of cells with a given phenotype is of great interest in diverse fields. A recent study explored three cell counting applications for viability determination for cultured cell lines, platelet counts in whole, unlysed human peripheral blood samples, and B- and T-cell concentrations in human peripheral blood. The study showed that a flow cytometer with traditional laminar flow fluidics and direct volume measurement capabilities is equally accurate and more precise, than either hemacytometer or counting bead methods for determining cell concentration.4 Not surprisingly, hemacytometer counts, including trypan blue for viability assessment, had the largest variability between quadruplicate counts on the same sample (average CV = 19.5%). The combined use of counting beads and the flow cytometer improved counting precision (average CV = 2.94%) over the hemacytometer method. However, the most precise measurement was obtained by direct volume measurement with the flow cytometer (average CV = 2.1%), with p values of 0.002 and 0.010 when compared to the hemacytometer and counting bead methods, respectively (Figure 4).
Conclusion Multi-parametric flow cytometric analysis can now replace other less accurate, time-consuming techniques for individual cell analysis. The new generation of two-laser, six-detector, compact flow cytometers successfully address the mainstream cell biology applications with a single platform, free up valuable bench space by reducing the need for application-specific instrumentation and make this valuable technique accessible to all life science researchers.
References 1. Vines, A. Blanco Fernández and G. McBean (2009). A Flow Cytometric Method for Continuous Measurement of Intracellular Calcium. Irish Flow Cytometry Meeting Poster.
2. Galbraith DW (2009). Simultaneous flow cytometric quantification of plant nuclear DNA contents over the full range of described angiosperm 2C values.
Cytometry 75A:692-698.
3. Chan SM, Olson JA, and Utz PJ (2005) Single-Cell Analysis of siRNA-Mediated Gene Silencing Using Multiparameter Flow Cytometry.
Cytometry 69A:59–65.
4. Rogers C., Dinkelmann M., Bair N., Rich C., Howes G., Eckert B. (2009) Comparison of Three Methods for the Assessment of Cell Phenotype, Viability, and Concentration in Cultures and Peripheral Blood. ASCB Poster.
About the Authors
This article was a collaborative effort by many individuals at Accuri Cytometers including MaryAnn Labant MS, MBA, Communications Manager, Maria Dinkelmann PhD, Laboratory Manager, Clare Rogers MS, Application Scientist and Grant Howes, VP Marketing.
This article was published in Bioscience Technology magazine: Vol. 34, No. 3, March, 2010, pp. 14-16.