The Essential Cell Analysis Tool

Transcription

1 The Essential Cell Analysis Tool The Accuri C6 Flow Cytometer System represents a dramatic step forward in flow cytometry and cell analysis. For the first time, researchers have access to a high capability analytical cytometer at an affordable price. Sized to fit on any benchtop, the C6 Flow Cytometer System makes a powerful research tool available to a much wider range of life scientists. Accuri s user-driven design has resulted in a system that offers both performance and simplicity. Combined with the intuitive CFlow software, Accuri s product makes flow cytometry equally accessible to experts and new users alike. This article examines a variety of biological applications that take advantage of the Accuri C6 s cellular analysis capabilities.

2 Today s Life Science Research Conundrum 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 not only devours time but also 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 benchtop space. The ideal circumstance would be to have an instrument, which could perform a variety of multi-parametric applications. The essential cell analysis tool, the Accuri C6 Flow Cytometer System (Figure 1), is just that instrument. With a footprint, weight and price similar to a real-time qpcr system or microplate reader, the Accuri C6 puts an essential cell analysis tool into the hands of more biologists than ever before. The instrument offers all the performance features of larger, complex flow cytometers, and combined with the straightforward CFlow system software, does not require expert operators or dedicated staff, making flow cytometry equally available to both novices and experts. In this paper we will examine a variety of biological applications that take advantage of the Accuri C6 s cellular analysis capabilities. Figure 1. The Accuri C6 Flow Cytometer System Beyond Classical Immunophenotyping Life science researchers have used flow cytometry for an expanding set of cell analysis applications over the past forty years. This quantitative analytical technology is often used to study cellular phenomena that might also be investigated by fluorescence microscopy, imaging or microplate reading. Beyond classical immunophenotyping, 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. Interest in flow cytometry has been further boosted with the current growth in proteomics and high throughput cell-based drug screening as well as the increasing use of biomarkers in drug development. TM Flow cytometry within reach. 1

3 The Issues 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. Due to the wide variations in fluorescence and light scatter signals produced by different types of cells and particles, flow cytometrists often spend significant time, and waste significant amounts of sample, optimizing amplifier gains and/or PMT voltage settings. These settings cannot be changed post-analysis if the cytometrist determines that signals were improperly amplified. This can result in the irretrievable loss of data. The Accuri C6 Flow Cytometer is equipped with pre-optimized detectors calibrated to operate within their linear range, so it 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. The combination of high-resolution digital-signal processing (24-bit DSP), resulting in more than six decades of dynamic range on all detectors, and the software capability to zoom in on very small areas of data display make the Accuri C6 easy to use for both novices and experienced flow cytometrists (Figure 2). Figure 2. Using the Zoom Tool in CFlow software with fixed permeabilized human peripheral blood. 24-Bit Digital Processing Provides Unique Advantages One of the first experiments we Many users have found that the 24-bit digital signal processing of the Accuri C6 Flow Cytometer provides major advantages for ease of data acquisition. The universal problem of setting and optimizing the detector gain in order to accommodate the diversity of fluorescence intensities from a wide range of samples is eliminated. Gating strategies and fluorescence compensation values can be set during, or after, data collection. Data collected on the Accuri C6 is never lost and can be re-analyzed at any time if gating or compensation errors are discovered, or in light of new research findings. ran was to establish the range of the 24-bit processor. A GFP signal that was off scale on our other flow cytometer gave us a distribution that was contained within the 24-bit scale on the Accuri C6. - Flow Cytometry Core Facility Manager 2

4 Varied Biological Applications Fluorescence microscopy, microplate reading, qpcr and Western blots are frequently used for a variety of cell studies. The Accuri C6 Flow Cytometer offers several unique advantages by enabling multi-parametric, individual-cell analysis. By incorporating a linear dynamic range greater than 6 decades, the Accuri C6 quantitatively captures the entire scope of biological variations in a single run without the need for data acquisition optimization or tuning. The Accuri C6 can analyze single to hundreds of thousands of individual cells, from heterogeneous populations, in hundreds of samples, with unattended operation. Rare events involving just one cell in ten thousand cells can be flagged. The Accuri C6 counts up to 10,000 events per second and up to 1 million events per sample. Another powerful feature of the C6 is its ability to quantitatively measure the concentration of cells or particles in samples. Absolute cell counts are correlated with the specific, known volumes being sampled. Just 18 inches wide and 17 inches deep, (36.3 x 41.9 cm), the 30-pound (13.6 kg) Accuri C6 Flow Cytometer fits on any lab bench or within a biological safety cabinet and can be easily transported. The Accuri C6 makes it possible to perform experiments on demand. The Accuri C6 provides important additional data that we have previously been missing. One is the highly accurate volume determination, so that absolute cell counts are automatically recorded for each experiment. Secondly, the six-log scale means that we can collect all data without compensation, so that runs from different days can be more easily compared. - Associate Professor of Immunology and Pediatrics We will now examine how C6 flow cytometry can improve outcomes from six existing techniques Western Blots GFP Transfection Studies Continuous Measurement of Intracellular Ca 2+ Cell Cycle Analysis and Ploidy RNAi Knockdown Cell Counting TM Flow cytometry within reach. 3

5 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 (Figure 3). 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. In addition flow cytometry can be used to investigate whether the permeabilization procedure used for targeting intracellular apoptosis-related proteins influences the profiles of cell surface antigen expression needed to identify MRD cells. Figure 3. Detection of sirna-mediated protein knockdown with flow cytometry, Western blotting, and immunofluorescence microscopy. (A) Density plot of Jurkat T cells 48 h post-transfection with either a non-specific control or Lck-specific pool of sirna. Forward scatter (FSC-H) is on the X-axis and intracellular Lck staining on the Y-axis. A representative gate circumscribing the Lcklo subpopulation is shown. Histograms of the corresponding cell populations for Lck are shown in the insets (green histograms). Red histogram plots represent background staining with purified rabbit gamma globulin. These plots are representative of ten independent experiments. (B) Western immunoblot of two nonspecific control sirna samples (lanes 1 and 2) and three Lckspecific sirna samples (lanes 3 5). Equal amount of protein was loaded in each lane. Samples were prepared from Jurkat cells 48 h post-transfection. The blot was probed with the same Lck antibody used for flow cytometry. (C) Immunofluorescence images of Jurkat cells transfected with control sirna or Lck sirna. Transfected cells were fixed, permeabilized, and stained with the same Lck antibody used in flow cytometry. A FITCconjugated donkey anti-rabbit (DAR) antibody was used for secondary detection. Cells were also stained with DAPI to reveal the location of nuclei. White arrows point to two cells with diminished Lck staining. Topleft image represents background staining with nonspecific rabbit gamma globulin (RGG)

6 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 fluorochromeconjugated 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. As opposed to bulk detection approaches examining the whole well in a microplate, flow cytometry analysis of individual viable cells can pick up very rare events and can find rare subpopulations. Continuous Measurement of Intracellular Ca 2+ 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, such as the Accuri C6, 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 (Figure 4). 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. With the Accuri C6, dynamic Ca2+ concentration measurement becomes extremely accessible and accurate, while also providing extensive and valuable data regarding population health and responsiveness. A major advantage of this system is that continuous monitoring of thousands of cells is possible, providing a method for highly accurate dynamic Ca 2+ measurements of the entire population. - Flow Cytometry Core Facility Manager Figure 4. Dot plots of continuous calcium dynamics, clearly indicating the absence of gaps on addition of test compounds. Fluorescence of Fluo-4 and Forward Scatter versus time. 2 Positive Control EGTA A23187 EGTA TG A23187 EGTA 2-APB TG A23187 TM Flow cytometry within reach. 5

7 Cell Cycle Analysis and Ploidy Biological experiments from the plant kingdom illustrate several key performance features of the Accuri C6 Flow Cytometer. 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 over 6 decades of digital signal linearity, the Accuri C6 Flow Cytometer 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. With the high degree of reproducibility, the requirement for using internal standards may be lessened, over time. 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 5). Accuri C6 Flow Cytometer data shows a near-perfect linear correlation of the data with r2= and %CVs for the first four peaks of about 3%. Day-to-day reproducibility of the 2C peak is about 7%. Figure 5. Flow cytometric analysis of homogenates prepared from A. thaliana root tissues. A B C 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

8 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. Since flow cytometry is multi-parametric, fluorescent antibodies can be used to not only assess the sirnatargeted 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 (Figure 6). Figure 6. Simultaneous detection of sirna-mediated protein down-regulation and functional responses to stimulation using multiparameter flow cytometry. Jurkat T cells were transfected with Lck sirna and 48 h later, stimulated with PHA-L at 5 lg/ml for 16 h in complete RPMI. Cells were then dually stained for CD69 up-regulation and Lck expression. Red histograms represent CD69 expression on either Lckhi (left panel) or Lcklo (right panel) gated subpopulations. Blue histogram represents basal CD69 staining on unstimulated Jurkat cells. MFI denotes mean fluorescent intensity. This experiment is representative of three independent experiments. 1 TM Flow cytometry within reach. 7

9 Cell Counting The ability to quickly and precisely deter mine the absolute number or concentration of cells with a given pheno type is of great interest in diverse fields including clinical research and diagnostics, drug discovery, and cell biology. The compact, fully-digital Accuri C6 Flow Cytometer combines the advantages of hydro-dynami cally focused cell sampling, high data acquisition rates and excellent light scatter resolution with the ability to calculate absolute counts for any identified population in a sample. A recent study explored three cell counting applications for viability determi nation for cultured cell lines, platelet counts in whole, unlysed human peripheral blood samples, and B- and T-cell concentrations in human peripheral blood (Figure 7). The study showed that the Accuri C6 with traditional laminar flow fluidics and direct volume measurement capabilities, is equally ac curate and more precise, than either hemacytometer or counting bead methods for determining cell concentration. 4, 5 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 Accuri C6 improved counting precision (average CV = 2.94%) over the hemacytometer method. However, the most precise measurement was obtained by direct volume measurement with the Accuri C6 (average CV = 2.1%), with p values of and when compared to the hemacytometer and counting bead methods, respectively (Figure 8). Counting Bead: cell count per ml 10,000,000 1,000, ,000 10,000 1,000 y = 1.116x r² = n = 75 1,000 10, ,000 1,000,000 10,000,000 Direct Volume: cell count per ml sample Figure 7. Comparison of cell concentration measurements by two methods. The x axis represents cell concentration calculated by absolute cell counts in relation to volume sampled. The y axis represents cell concentration as calculated by the absolute cell counts in re lation to the number of SPHERO AccuCount beads measured. 75 different samples were analyzed in five independently run experiments. Cell types analyzed included human peripheral blood sam ples and mammalian cell lines Coefficient of Variation direct-volume (C6 cytometer) counting beads (C6 cytometer) p=0.010 Hemacytometer p=0.002 Figure 8. 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 =2.10%; counting bead method =2.94%; hemacytometer =19.51%) Cell Counting Method 8

10 The Must-Have Instrument for Every Biology Lab The Accuri C6 Flow Cytometer is the essential cell analysis tool for every biology lab. Multi-parametric flow cytometric analysis can now replace other less accurate, time-consuming techniques. By designing a 2-laser, 6-detector, compact flow cytometer, Accuri has successfully addressed the mainstream cell biology applications with a single platform and freed up valuable bench space by reducing the need for application-specific instrumentation. Life science researchers can put an Accuri C6 Flow Cytometer in their lab, along with a five-year extended warranty, for approximately the same cost as a 4-year service contract of a legacy flow cytometry system. The Accuri C6 Flow Cytometer offers flexibility with three levels of sample throughput capabilities: samples per hour with manual operation, samples per hour with the optional, automated CSampler (pictured below) and 384 samples in 10 minutes with the IntelliCyt TM HyperCyt high-throughput autosampler. I did not want another complicated piece of instrumentation to learn and maintain. As it turned out the Accuri C6 is not a complex instrument, is easy-to-use and it is also easy to train new users on. The C6 combination is an excellent addition to the core facility and gives us the capability to provide cellular and bead-based data. - High Throughput Screening, sirna, High Content Screening Laboratory Director Virtually any tube may be used with the Accuri C6 Flow Cytometer, from 12 x 75 mm test tubes to microfuge tubes to multi-well plates, reducing operational costs even further. Reagents can be easily added to samples during data acquisition to study cell processes dynamically. Affordable, accessible flow cytometry is now a reality for all biology researchers. The Accuri C6 puts an essential cell analysis tool into the hands of more biologists than ever before and offers all the performance features of larger, complex flow cytometers making it the platform of choice for multiparametric cell analysis experiments. TM Flow cytometry within reach. 9

11 References 1. Chan SM, Olson JA, and Utz PJ (2005) Single-Cell Analysis of sirna-mediated Gene Silencing Using Multiparameter Flow Cytometry. Cytometry 69A: Vines, A. Blanco Fernández and G. McBean (2009). A Flow Cytometric Method for Continuous Measurement of Intracellular Calcium. Irish Flow Cytometry Meeting Poster. 3. Galbraith DW (2009). Simultaneous flow cytometric quantification of plant nuclear DNA contents over the full range of described angiosperm 2C values. Cytometry 75A: 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. 5. Genetic Engineering News (2009). Cell-Counting Technologies Are Evolving. Vol. 29, No. 17: Ordering Information and Technical Support In the U.S., call: In Europe, call: +44 (0) For other countries, go to: Accuri Cytometers, Inc. 173 Parkland Plaza Ann Arbor, MI USA CustomerService@AccuriCytometers.com Accuri Cytometers (Europe) Ltd 56 Edison Road St. Ives Cambs PE27 3LF UK EuroTech@AccuriCytometers.com 2010 Accuri Cytometers, Inc. Accuri, C6 Flow Cytometer, CSampler, and CFlow are registered trademarks of Accuri Cytometers, Inc.